Transferrin-binding proteins from n.gonorrhoeae and n. meningitidis

ABSTRACT

Iron-regulated, outer membrane proteins found in  Neisseria gonorrhoeae  and  Neisseria meningitidis  are important in transferrin receptor function. The proteins, which are isolatable by means of a transferrin affinity column, bind specifically to antisera raised against an iron-regulated outer membrane protein having a molecular weight of approximately 100 kD found in  Neisseria gonorrhoeae.

[0001] This specification is a continuation-in-part of Ser. No. 07/572,187 filed Aug. 23, 1990, which is incorporated herein by reference.

[0002] The invention disclosed in the specification is directed to transferrin-binding proteins from Neisseria gonorrhoeae and Neisseria meningitidis as well as immunologically cross-reactive fragments and analogs thereof. The specification is further directed to antibodies raised against such proteins, as well as the use of such proteins and antibodies in the detection of N. gonorrhoeae and N. meningitides and treatment of diseases caused by N. gonorrhoeae and N. meningitidis. DNA encoding recombinant transferrin-binding proteins and cells that express such DNA are also covered by the present invention.

[0003]N. gonorrhoeae and N. meningitidis are two pathogens of the genus Neisseria that are genetically similar, but pathogenically different. Iron is an essential nutrient for the growth of N. gonorrhoeae and N. meningitidis, as it is for many bacteria. Unlike most other gram negative bacteria, N. gonorrhoeae and N. meningitidis do not produce and secrete small, soluble iron-chelating compounds, called siderophores. These other gram-negative bacteria have receptors capable of taking up the iron-siderophore complex.

[0004] Instead, N. gonorrhoeae and N. meningitidis are believed to possess membrane proteins that bind to the iron-binding glycoproteins lactoferrin and transferrin, which are present in human exocrine secretions and serum, respectively. N. gonorrhoeae and N. meningitidis are believed to take up iron in human hosts through the binding of lactoferrin and transferrin to these lactoferrin- and transferrin-binding membrane proteins, i.e, receptors.

[0005] The lactoferrin-binding protein from N. meningitidis is believed to be a 105kD, iron-regulated outer membrane protein; see Schryvers and Morris, Infect. Immun. 56, 1144-1149 (1988). The transferrin-binding protein from one strain of N. meningitidis has been reported to be a 71 kD iron-regulated outer membrane protein, although other strains are reported to have transferrin-binding proteins with molecular weights of 75 kD-88 kD, 85 kD, and 95 kD; see Schryvers and Morris, Mol. Microbiol. 2, 281-288 (1988). These authors concede that the results of the various attempts at identifying the transferrin-binding protein of N. meningitidis are not consistent with each other. In fact, proteins of 85 kD and 95 kD are shown not to be necessary for transferrin receptor function in N. meningitidis; see Dyer et al., Microbial Pathogenesis 3, 351-363 (1987).

[0006] The ability of N. gonorrhoeae to assimilate iron has also been of interest. In one investigation, a dot binding assay involving the use of gonococcal total membranes derived from cells grown under iron-deficient conditions suggested the presence of separate receptors for lactoferrin and transferrin. The molecular weight and other properties of the binding proteins are not determined. See Lee and Schryvers, Mol. Microbiol. 2, 827-829 (1988). Therefore, the identity of the binding proteins in N. gonorrhoeae has not previously been established.

[0007] The diseases caused by gonococcal and meningococcal infection are pervasive and often serious. Improved methods for preventing, detecting and treating such diseases, such as gonorrhea, meningitis and septic shock are needed.

[0008] The growth of N. gonorrhoeae and N. meningitidis in humans can be inhibited by reducing the ability of these cells to take up iron. A reduction in the ability of gonococcal and meningococcal cells to assimilate iron in the bloodstream could be accomplished by blocking the transferrin receptor function. The transferrin receptor, for example, could be blocked by antibodies against the receptor. In order to raise antibodies against the receptor, however, the receptor must be identified so that it can be isolated.

[0009] There is, therefore, a need for identifying, isolating and purifying the transferrin-binding proteins from N. gonorrhoeae and N. meningitidis. DNA molecules encoding such proteins are needed in order to produce recombinant transferrin binding proteins. Antibodies against the transferrin binding proteins are needed in order to inhibit transferrin receptor function. Vaccines are needed to prevent and to treat gonococcal and meningococcal infections. Antibody and nucleic acid probes are needed to detect N. gonorrhoeae and N. meningitidis. It is the object of the present invention to provide such proteins, antibodies, DNA molecules and vaccines for detecting, preventing and treating gonococcal and meningococcal infections.

SUMMARY OF THE INVENTION

[0010] These and other objectives as will become apparent to those having ordinary skill in the art have been achieved by providing an iron-regulated protein found in Neisseria gonorrhoeae or Neisseria meningitidis outer membranes,

[0011] wherein the protein is substantially free of:

[0012] (a) detergent;

[0013] (b) nitrocellulose/cellulose acetate paper; and

[0014] (c) other iron-regulated proteins from Neisseria gonorrhoeae and Neisseria meningitidis;

[0015] wherein the protein is isolatable by means of a transferrin affinity column;

[0016] wherein the protein binds specifically to antisera raised against an iron-regulated outer membrane protein having a molecular weight of approximately 100 kD found in Neisseria gonorrhoeae; and

[0017] wherein the protein is important in transferrin receptor function in Neisseria gonorrhoeae or Neisseria meningitidis; and functional analogs of such proteins.

[0018] The invention further provides DNA molecules that express the transferrin binding protein and its analogs in a host cell. The resulting recombinant protein is also part of the invention.

[0019] The invention also includes antibodies against the transferrin-binding proteins of the invention. The antibodies inhibit growth of N. gonorrhoeae and/or N. meningitidis, and are useful in controlling infections of these pathogens.

[0020] The invention further includes vaccine compositions comprising the transferrin-binding proteins of the invention and analogs of such proteins, as well as methods of immunizing a host against gonococcal and meningococcal diseases, such as gonorrhea, meningitis, and septic shock, by administering such vaccines. The antibodies of the invention may be used in passive immunization to treat gonococcal and meningococcal diseases.

DESCRIPTION OF THE FIGURES

[0021]FIG. 1 shows the entire DNA and amino acid sequence encoding the 100 kD gonoccocal transferrin binding protein 1 (TBP1). The start codon encoding the first amino acid of the mature protein occurs at nucleotide 406. The stop codon for the protein occurs at nucleotide 3153. See example 6a and example 7. See SEQ ID NO:1 and SEQ ID NO:2.

[0022]FIG. 2 represents transferrin binding protein 1 clones from which the entire gene sequence for the protein is derived.

[0023]FIG. 3 shows positions of transposon insertions within the 100 kD gonococcal transferrin binding protein fragment in pUNCH403 and corresponding phenotypes of respective mutants. Transposons (mTn3CAT) are inserted by shuttle mutagenesis in E. coli. Chloramphenicol resistant transformants are selected in FA19 to create mutants. Below each transposon insertion (indicated by inverted triangle), growth on 2.5 μM human transferrin (25% saturated with Fe) and expression of protein as assayed by Western blot are indicated by +or −. The open reading frame, indicated by an arrow, reads right to left and begins with methionine, designated M. A typical −10 sequence was found (−10) but no canonical −35 sequence could be identified. Wild-type growth and protein expression are shown at right under the heading “No Tn”. See example 9.

[0024]FIG. 4 shows a strategy for cloning the meningococcal 95 kD transferrin binding protein gene. The figure is not drawn to scale. The 1.3 kb HincII/EcoRI fragment shown in step 1 is cloned from a lambda Zap II library using the anti-100 kD protein antibody probe described in Example 4. The method for screening the library is described in Example 6a. The 5.0 kb fragment shown in step 2 is cloned from a partial ClaI library in pHSS6-GCU using the 1.3 kb fragment as a probe. The 2.0 kb EcoRI/HincII fragment in step 3 is cloned from a lambda Zap II library using the 1.7 kb EcoRI/ClaI restriction fragment from step 2 as a probe. The 2.5 kb EcoRI/HincII fragment shown in step 4 is cloned from a lambda Zap II library using the 2.0 kb EcoRI/HincII fragment from step 3. The fragments from steps 1-4 fit together as shown in the fragment entitled “FAM20 Chromosome”. See example 10.

[0025]FIG. 5 shows the entire DNA and amino acid sequence of the 95 kd meningococcal transferrin-binding protein. The ATG start codon encoding the first amino acid of the mature protein occurs at nucleotide 721. The stop codon is at nucleotide 3450. See example 10. See SEQ ID NO:3 and SEQ ID NO:4.

[0026]FIG. 6 shows the results of transposon mutagenesis experiments involving the 1.3 kb HincII/EcoRI fragment from step 1 of FIG. 4. See example 11.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Isolation of Proteins from Bacteria

[0028] Transferrin-binding proteins are prepared from the membranes of N. gonorrhoeae or N. meningitidis. The membranes may be prepared by methods known in the art. The method described by Schryvers and Morris in Infect. Immun. 56, 1144-1149 (1988) is suitable. This method is incorporated herein by reference.

[0029] The membranes are obtained from cells grown in an iron-deficient medium. The growth medium may be a standard growth medium such as GC medium base (gonococcal medium base) supplied by Difco. This medium can be made iron-deficient by the addition of chelating agents such as ethylenediaminetetraacetic acid (EDTA), ethylene-diamine-di-ortho-hydroxyphenylacetic acid (EDDA), or desferal (Ciba Pharmaceuticals). Alternatively, the growth medium may be a chemically defined medium described by Mickelsen and Sparling (Inf. Immun. 33, 555-564 (1981)), which is made iron-deficient by treatment with the chelating agent Chelex-100 (Bio-Rad).

[0030] Any gonococcal and meningococcal strains that have normal transferrin receptor function are useful in the present invention. Such strains are generally available from clinical and other sources, such as the American Type Culture Collection, Bethesda, Md. and the Neisseria Repository, NAMRU, University of California, Berkley.

[0031] For example, gonococcal strains FA19, which is described in McKenna et al, Infect. Immun. 56, 785-791 (1988); FA248, which is described in Biswas et al, J. Bacteriol. 151, 77-82 (1979); and F62, which is described in West and Sparling, Infect. and Immun. 47, 388-394 (1985) constitute suitable sources of the gonococcal transferrin protein. Meningococcal strains FAM18 and FAM20 (Dyer et al., Microbial Pathogenesis 3, 351-363 (1987)) and B16B6, group X and group W135 (Schryvers and Morris 56, 1144-1149 (1988)) are representative of sources of the meningococcal transferrin binding protein.

[0032] Proteins that bind to transferrin may be isolated from other membrane proteins of iron-starved N. gonorrhoeae and N. meningitidis with immobilized transferrin using affinity procedures known in the art; see, for example, Schryvers and Morris, Infect. Immun. 56, 1144-1149 (1988). The method of Schryvers and Morris is incorporated herein by reference. A variation of this procedure, which is described in Example 2a, is preferably used to resolve the transferrin binding proteins from gonococcal and meningococcal, membrane proteins.

[0033] Briefly, membranes from iron-starved gonococcal and meningococcal cells are isolated and treated with biotinylated transferrin. The resulting complex is immobilized by, for example, treating the complex with avidin- or streptavidin-agarose. The affinity resin pellet is thoroughly washed and suspended in buffer. The transferrin receptor is separated from the immobilized transferrin by, for example, heating. The proteins are separated by, for example, SDS-PAGE in accordance with the method of Laemmli, Nature 227, 680-685 (1970). A protein having a molecular weight of approximately 100 kD, hereinafter 100 kD protein, is resolved from gonococci. A protein having a molecular weight of approximately 95 kD, hereinafter, 95kD protein, is resolved from meningococci.

[0034] Identification of Proteins

[0035] The molecular weights are determined by resolving single bands on SDS-PAGE and comparing their positions to those of known standards. The method is understood by those in the art to be accurate within a range of 3-5%. The molecular weights varied slightly between determinations. The molecular weight of the protein from gonococci is consistently and repeatably higher than that from meningococci, and varied from 97-100 kD.

[0036] Confirmation that the 100 kD transferrin-binding protein from N. gonorrhoeae is important for transferrin receptor function is obtained by preparing five different gonococcal mutants deficient in transferrin receptor activity. Each mutant is tested for the presence of the 100 kD transferrin-binding protein by western blot using polyclonal antisera raised in rabbits. In each mutant, the amount of 100 kD outer membrane protein is much less than is observed for wild-type gonococcal strains. Other mutant gonococcal strains that have normal transferrin receptor activity had wild-type levels of the 100 kD protein in their membranes.

[0037] A similar experiment establishes that the 95 kD protein from meningococci is important for transferrin receptor function. The western blot analysis is performed with antisera raised against the 100 kD protein from N. gonorrhoeae, which is found to cross-react with the 95 kD protein from N. meningitidis. Thus, in both N. gonorrhoeae and N. meningitidis, the lack of transferrin receptor activity correlates with the absence of the 100 kD and 95 kD proteins, respectively.

[0038] Therefore, contrary to expectations based on the prior art, the iron-regulated 100 kD outer membrane protein found in N. gonorrhoeae is the transferrin receptor. The iron-regulated 95 kD outer membrane protein found in N. meningitidis surprisingly cross-reacts with antisera raised against the 100 kD protein found in N. gonorrhoeae, and is the N. meningitidis transferrin receptor. Antisera raised in mammals, such as rabbits, mice, goats, monkeys and humans, against the transferrin receptor from N. gonorrhoeae are generally cross-reactive with the transferrin receptor from N. meningitidis and vice versa. Monoclonal antibodies are also generally cross-reactive with the 95 kD and 100 kD proteins.

[0039] As used herein, transferrin receptor from N. gonorrhoeae and N. meningitidis include the iron-regulated 100 kD outer membrane protein from N. gonorrhoeae and the iron-regulated 95 kD outer membrane protein from N. meningitidis. It should be understood that these transferrin receptors constitute a class of proteins. The class includes, for example, variations in the amino acid sequence that occur naturally in the various strains of N. gonorrhoeae and N. meningitidis.

[0040] The proteins of the present invention further include functional analogs of the 100 kD or the 95 kD transferrin receptors from N. gonorrhoeae or N. meningitidis, respectively. A protein is considered a functional analog of another protein for a specific function, as described below, if the analog is immunologically cross-reactive with, and has the same function as, the other protein. The analog may, for example, be a fragment of the protein, or a substitution, addition or deletion mutant of the protein.

[0041] The proteins and functional analogs of the present invention are essentially pure. For the purposes of this specification, essentially pure means that the proteins and functional analogs are free from all but trace amounts of other iron-regulated proteins from N. gonorrhoeae and N. meningitidis as well as of materials used during the purification process. The other iron-regulated proteins from N. gonorrhoeae and N. meningitidis include other transferrin binding proteins. Materials used in the purification process include detergents, affinity binding agents and separation films. Detergents include sodium dodecyl sulfate and sarcosine. Affinity binding agents include agarose, avidin-agarose, streptavidin-agarose, biotin and biotinylated proteins, such as biotinylated transferrin. Separation films include nitrocellulose paper and nitrocellulose/cellulose acetate paper.

[0042] Recombinant DNA

[0043] Methods are known for isolating DNA once the protein has been isolated and purified. Many of these methods are described in Maniatis et al, “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press (1982). The immunological screening method is preferred.

[0044] For example, chromosomal DNA from a gonococcal or meningococcal strain capable of utilizing iron bound to transferrin, such as those described above, is isolated and cleaved into fragments of suitable size by standard methods. Suitable DNA cleavage methods include, for example, sonication and the use of restriction endonucleases. A suitable average fragment size is approximately 0.5-10 kbp.

[0045] Linkers are added to the fragments and the resulting fragments are ligated into a suitable vector. The linker corresponds to a restriction site in the vector. Suitable linkers include, for example, EcoRI, PstI and BamHI. A suitable vector is lambda-gt11. Ligated DNA may be packaged by commercial kits, such as a kit manufactured by Promega.

[0046] Proteins from the resulting library are cloned and expressed in a suitable host, typically E. coli. Cloning is preferably performed in an E. coli host carrying the following mutations: mcrA, mcrB, mcrC, mrr, hsdS, hsdr, and hsdM. Some suitable E. coli strains include DH5alphaMCR (BRL) and “SURE” (Stratagene).

[0047] The plaques that are obtained are screened immunologically by methods known in the art. Maniatis, Id. A suitable method is described in Example 6 below. Screening may be facilitated by the use of a commercial screening kit, such as the Picoblue Immunological Screening Kit of Stratagene (La Jolla, Calif.) in accordance with the accompanying Stratagene protocol, which is available from Stratagene or from the file history of this specification.

[0048] Plaques that bind the transferrin-binding protein specific antisera are selected from non-reacting plaques and purified. Maniatis, Id. The DNA from purified phage is isolated by methods known in the art. Suitable methods include, for example, polyethylene glycol precipitation, phage lysis, and anion exchange chromatography, which can be facilitated by the use of a kit manufactured by Qiagen (Studio City, Calif.).

[0049] The DNA obtained may be amplified by methods known in the art. One suitable method is the polymerase chain reaction (PCR) method described by Mullis et al in U.S. Pat. No. 4,683,195 and by Sambrook, Fritch and Maniatis (eds) in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). It is convenient to amplify the DNA clones in the lambda-gt11 vectors using lambda-gt11-specific oligomers available from New England Biolabs.

[0050] Amplified clones are inserted into suitable vectors and sequenced in accordance with methods known in the art. A suitable sequencing method is the dideoxy chain terminating method described by Sanger et al. in Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977).

[0051] Suitable vectors and polymerases for sequencing are known. A suitable vector is the Bluescript vector of Stratagene. A suitable polymerase is Sequenase (United States Biochemical Corp., Cleveland, Ohio).

[0052] In the immunoscreening method described above, it is usually necessary to screen a large number of plaques in order to identify fragments with the transferrin-binding protein specific antisera. For example, in one experiment, approximately 500,000 plaques are obtained from fragments of a gonococcal (FA19) chromosome. Two plaques are identified using the antisera against the 100 kD transferrin-binding protein from N. gonorrhoeae. A clone having an insert size of 323 bp (pUNCH401) is isolated from one plaque, while a clone with an insert size of 483 bp (pUNCH402) is isolated from the other plaque. These DNA sequences represent overlapping fragments of the FA19 chromosome. The consensus sequence of the two fragments, including the overlap, is shown as FIG. 1. Nucleotides 75 to 323 represent the overlapping sequences. Nucleotides 1 to 74 represent the non-overlapping sequence of the 323 bp fragment. Nucleotides 324 to 558 represent the non-overlapping sequence of the 483 bp fragment. The only open reading frame runs in the direction opposite to that shown in FIG. 1 (i.e. from nucleotide 558 to nucleotide 1). See example 6a.

[0053] The fragments described above, or sub-fragments of them, can be used as probes for obtaining additional fragments of the transferrin-binding protein gene. Using this technique, an 8 kb ClaI fragment and a 3.2 kb HincII fragment in the FA19 chromosome hybridizes to the 323 and 483 bp fragments. A restriction map of the 3.2 kb HincII fragment is shown in FIG. 2. Fragments obtained can be sequenced. See examples 7 and 8.

[0054] By suitable extensions of the fragments, the entire gene is sequenced. The limits of the coding sequence are determined by methods known in the art, such as by insertional mutagenesis. See example 9. Similar methods are used to determine the sequence of the 95 kD meningococcal transferrin binding protein. See examples 10 and 11 and FIGS. 4-6.

[0055] Recombinant Proteins

[0056] The proteins of the present invention may be produced by means of recombinant DNA technology. Suitable methods for producing recombinant proteins from isolated DNA are described in Maniatis et al., Id.

[0057] Briefly, DNA coding for the transferrin-binding proteins of the present invention, as well as DNA coding for their functional analogs, may be expressed using a wide variety of host cells and a wide variety of vectors. The host may be prokaryotic or eukaryotic. The DNA may be obtained from natural sources and, optionally, modified. The DNA may also be synthesized in whole or in part.

[0058] The vector may comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences. Some suitable prokaryotic vectors include plasmids from E. coli such as colE1, pCR1, pBR322, PMB9, and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages.

[0059] Vectors useful in yeast are available. A suitable example is the 2μ plasmid.

[0060] Suitable vectors for use in mammalian cells are also known. Such vectors include well-known derivatives of SV-40 adenovirus, retrovirus-derived DNA sequences and vectors derived from combination of plasmids and phage DNA.

[0061] Further eukaryotic expression vectors are known in the art (e.g., P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S. Subramani et al, Mol. Cell. Biol. 1, 854-864 (1981); R. J. Kaufmann and P. A. Sharp, “Amplification And Expression Of Sequences Cotransfected with A Modular Dihydrofolate Reductase Complementary DNA Gene,” J. Mol. Biol. 159, 601-621 (1982); R. J. Kaufmann and P. A. Sharp, Mol. Cell. Biol. 159, 601-664 (1982); S. I. Scahill et al, “Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,” Proc. Natl. Acad. Sci. USA 80, 4654-4659.(1983); G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA 77, 4216-4220, (1980).

[0062] Useful expression hosts include well-known prokaryotic and eukaryotic hosts. Some suitable prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRCl, Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces. Suitable eukaryotic cells include yeasts and other fungi, insect, animal cells, such as COS cells and CHO cells, human cells and plant cells in tissue culture.

[0063] The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the transferrin-binding protein gene or fragment thereof. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

[0064] The 100 kD or 95 kD proteins may be purified by methods known in the art. For example, the entire transferrin binding proteins or portions thereof may be expressed in the form of a fusion protein with an appropriate fusion partner. The fusion partner preferably facilitates purification and identification. Some useful fusion partners include beta-galactosidase (Gray, et al., Proc. Natl. Acad.

[0065] Sci. USA 79, 6598 (1982)); trpE (Itakura et al., Science 198, 1056 (1977)) and protein A (Uhlen et al., Gene 23 369 (1983)). For example, fusion proteins containing beta-galactosidase may be purified by affinity chromatography using an anti-beta-galactosidase antibody column (Ullman, Gene. 29, 27-31 (1984)).

[0066] It is preferable that the DNA that encodes the fusion protein is engineered so that the fusion protein contains a cleavable site between the transferrin binding protein and the fusion partner. Both chemical and enzymatic cleavable sites are known in the art. Suitable examples of sites that are cleavable enzymatically include sites that are specifically recognized and cleaved by collagenase (Keil et al., FEBS Letters 56, 292-296 (1975)); enterokinase (Hopp et al., Biotechnology 6, 1204-1210 (1988)); factor Xa (Nagai et al., Methods Enzymol. 153, 461-481 (1987)); thrombin (Eaton et al., Biochemistry 25, 505 (1986)); and glutathione S-transferase (Johnson, Nature 338, 585 (1989); and Van Etten et al., Cell 58, 669 (1989)). Collagenase cleaves between proline and X in the sequence Pro-X-Gly-Pro wherein X is a neutral amino acid. Enterokinase cleaves after lysine in the sequence Asp-Asp-Asp-Asp-Lys. Factor Xa cleaves after arginine in the sequence Ile-Glu-Gly-Arg. Thrombin cleaves between arginine and glycine in the sequence Arg-Gly-Ser-Pro.

[0067] Specific chemical cleavage agents are also known. For example, cyanogen bromide cleaves at methionine residues in proteins.

[0068] Alternatively, the 100 kD and 95 kD transferrin receptor proteins may be overexpressed behind an inducible promoter and purified by affinity chromatography using specific transferrin receptor antibodies. As another alternative, the overexpressed protein may be purified using a combination of ion-exchange, size-exclusion, and hydrophobic interaction chromatography using methods known in the art. These and other suitable methods are described by Marston, “The Purification of Eukaryotic Polypeptides Expressed in E. coli” in DNA Cloning, D. M. Glover, Ed., Volume III, IRL Press Ltd., England, 1987.

Utility

[0069] Proteins as Probes

[0070] The 100 kD protein from N. gonorrhoeae, the 95 kD protein from N. meningitidis, and their functional analogs are useful in detecting and preventing diseases caused by gonococcal and meningococcal infection.

[0071] For example, the proteins may be labelled and used as probes in standard immunoassays to detect antibodies against the proteins in samples, such as in the sera or other bodily fluids of patients being tested for gonorrhea, septic shock, or meningitis. In general, a protein in accordance with claim A or a functional derivative of such a protein is incubated with the sample suspected of containing antibodies to the protein. The protein is labelled either before, during, or after incubation. The detection of labelled protein bound to an antibody in the sample indicates the presence of the antibody. The antibody is preferably immobilized.

[0072] Suitable assays for detecting antibodies with proteins are known in the art, such as the standard ELISA protocol described by R. H. Kenneth, “Enzyme-Linked Antibody Assay with Cells Attached to Polyvinyl Chloride Plates” in Kenneth et al, Monoclonal Antibodies, Plenum Press, N.Y., page 376 (1981). Briefly, plates are coated with a sufficient amount of an antigenic protein to bind detectable amounts of the antibody. After incubating the plates with the polypeptide, the plates are blocked with a suitable blocking agent, such as, for example, 10% normal goat serum. The sample, such as patient sera, is added and titered to determine the endpoint. Positive and negative controls are added simultaneously to quantitate the amount of relevant antibody present in the unknown samples. Following incubation, the samples are probed with goat anti-human Ig conjugated to a suitable enzyme. The presence of anti-protein antibodies in the sample is indicated by the presence of the enzyme.

[0073] For use in immunoassays, the protein may be labelled with radioactive or non-radioactive atoms and molecules. Such labels and methods for conjugating them to proteins are known in the art.

[0074] Some examples of useful radioactive labels include ³²P, ¹²⁵I, ¹³¹I, and ³H. Use of radioactive labels have been described in U.K. 2,034,323, U.S. Pat. No. 4,358,535, and U.S. Pat. No. 4,302,204.

[0075] Some examples of non-radioactive labels include enzymes, chromophors, atoms and molecules detectable by electron microscopy, and metal ions detectable by their magnetic properties.

[0076] Some useful enzymatic labels include enzymes that cause a detectable change in a substrate. Some useful enzymes and their substrates include, for example, horseradish peroxidase (pyrogallol and o-phenylene-diamine), beta-galactosidase (fluorescein beta-D-galactopyranoside), and alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The use of enzymatic labels have been described in U.K. 2,019,404, EP 63,879, and by Rotman, Proc. Natl. Acad. Sci., 47, 1981-1991 (1961).

[0077] Useful chromophores include, for example, fluorescent, chemiluminescent, and bioluminescent molecules, as well as dyes. Some specific chromophores useful in the present invention include, for example, fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol.

[0078] The labels may be conjugated to the probe by methods that are well known in the art. The labels may be directly attached through a functional group on the probe. The probe either contains or can be caused to contain such a functional group. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.

[0079] The label may also be conjugated to the probe by means of a ligand attached to the probe by a method described above and a receptor for that ligand attached to the label. Any of the known ligand-receptor combinations is suitable. The biotin-avidin combination is preferred.

[0080] For use in immunoassays, the proteins may be the entire 100 kD or 95 kD protein or may be functional analogs thereof. Functional analogs of these proteins include fragments and substitution, addition and deletion mutations that do not destroy the ability of the proteins to bind to their antibodies. As long as the proteins are able to detect antibodies specific for the transferrin-binding proteins, they are useful in the present invention.

[0081] Proteins in Vaccines

[0082] Since the transferrin-binding proteins of the present invention are important for a vital function of N. gonorrhoeae and N. meningitidis, and are found on the outer membranes, these proteins are useful in vaccines for the prevention of diseases caused by Neisseria infections, such as gonorrhea, septic shock, and meningitis. For this purpose, it is necessary for the protein to produce neutralizing antibodies. Neutralizing antibodies are antibodies that significantly inhibit the growth of and/or kill the bacterial cells in vitro or in vivo. Growth of the bacteria is significantly inhibited in vivo if the inhibition is sufficient to prevent or reduce the symptoms of the disease of a mammal infected with the disease.

[0083] Vaccines comprising the 100 kD or 95 kD protein or functional analogs as antigen may be used to inhibit the growth of, or kill, the gonococci or meningococci in accordance with the invention. Functional analogs of the 100 kD and 95 kD proteins for this purpose include fragments and substitution, addition or deletion mutations that produce neutralizing antibodies in a mammalian host such as in a human host.

[0084] The present invention further includes vaccine compositions for immunizing mammals, including humans, against infection by N. gonorrhoeae and N. meningitidis. The vaccines comprise the 100 kD transferrin receptor from N. gonorrhoeae and/or the 95 kD transferrin receptor from N. meningitidis and pharmaceutically acceptable adjuvants. Instead of the 100 kD and 95 kD proteins, functional analogs may-be substituted, as described above.

[0085] The vaccine comprises the antigen in a suitable carrier. The vaccine may include adjuvants, such as muramyl peptides, and lymphokines, such as interferon, interleukin-1 and interleukin-6. The antigen may be adsorbed on suitable particles, such as aluminum oxide particles, or encapsulated in liposomes, as is known in the art.

[0086] The antigen may also be delivered in an avirulent strain of Salmonella, such as S. typhimurium. Such vaccines may be prepared by cloning DNA comprising the active portion of the transferrin binding protein in the Salmonella strain, as is known in the art; see, for example, Curtiss et al., Vaccine 6, 155-160 (1988) and Galan et al., Gene 94, 29-35 (1990).

[0087] The invention further includes methods of immunizing host mammals, including humans, with the vaccine compositions described above. The vaccine may be administered to a mammal by methods known in the art. Such methods include, for example, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.

[0088] The vaccine composition may contain the entire 100 kD protein or the 95 kD protein, but preferably contains a non-toxic fragment of the 100 kD or 95 kD protein. It is well known, for example, to produce fragments of antigenic proteins and to determine those fragments that contain the antigenic site. The length of the fragment is not critical as long as the fragment is antigenic and non-toxic. Therefore, the fragment should contain sufficient amino acid residues to define the epitope. Methods for isolating and identifying antigenic fragments from known antigenic polypeptides are described by Salfeld et al. in J. Virol. 63, 798-808 (1989) and by Isola et al. in J. Virol. 63, 2325-2334 (1989).

[0089] If the fragment defines the epitope, but is too short to be antigenic, it may be conjugated to a carrier molecule. Some suitable carrier molecules include keyhole limpet hemocyanin and bovine serum albumen. Conjugation may be carried out by methods known in the art. One such method is to combine a cysteine residue of the fragment with a cysteine residue on the carrier molecule.

[0090] Antibodies for Treatment

[0091] Further, the invention includes isolating neutralizing antibodies that specifically recognize and bind to the proteins and functional analogs of the invention. The antibodies may be polyclonal or monoclonal. The definitions of neutralizing antibodies and functional analogs used in conjunction with vaccines (see above) apply as well to the production of neutralizing antibodies

[0092] Polyclonal antibodies are isolated from mammals that have been innoculated with the protein or a functional analog in accordance with methods known in the art. The monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method of Kohler and Milstein, Nature 256, 495-497 (1975) and the recombinant DNA method described by Huse et al. in Science 246, 1275-1281 (1989).

[0093] The invention also includes methods of treating mammals, including humans, suffering from diseases caused by N. gonorrhoeae or N. meningitidis by administering to such mammals an effective amount of the neutralizing antibodies of the invention. Administration may be by the same methods described above for administering vaccines.

[0094] Antibodies as Probes

[0095] The transferrin-binding proteins and functional analogs of the invention may also be used to produce antibodies for use as probes to detect the presence of Neisseria gonorhoeae or Neisseria meningitidis in a sample. The antibodies may be polyclonal or monoclonal. For this purpose, functional analogs include fragments and substitution, addition and deletion mutations of the 100 kD protein or of the 95 kD protein as long as the analogs also produce antibodies capable of detecting the presence of the 100 kD or 95 kD proteins in a sample. The sample may, for example, be a bodily fluid from a mammal, including a human, suspected of being infected with N. gonorrhoeae or N. meningitidis.

[0096] Assays for detecting the presence of proteins with antibodies have been previously described, and follow known formats, such as standard blot and ELISA formats. These formats are normally based on incubating an antibody to a sample suspected of containing the 95 kD or 100 kD protein and detecting the presence of a complex between the antibody and the protein. The antibody is labelled either before, during, or after the incubation step. The protein is preferably immobilized prior to detection. Immobilization may be accomplished by directly binding the protein to a solid surface, such as a microtiter well, or by binding the protein to immobilized antibodies.

[0097] When used as probes, the antibodies are normally labelled by methods known in the art. The same labels useful for proteins (see above) are also useful for antibodies. Methods for labelling antibodies have been described, for example, by Hunter and Greenwood in Nature 144, 945 (1962) and by David et al. in Biochemistry 13, 1014-1021 (1974). Additional methods for labelling antibodies have been described in U.S. Pat. Nos. 3,940,475 and 3,645,090.

[0098] Nucleic Acid Molecules as Probes

[0099] Nucleic acid molecules encoding the 100 kD protein, the 95 kD protein, or fragments of the 100 kD or 95 kD proteins having unique sequences may be used to detect the presence of N. gonorrhoeae or N. meningitidis. The nucleic acid molecules may be RNA or DNA.

[0100] Methods for determining whether a nucleic acid molecule probe recognizes a specific nucleic acid molecule in a sample are known in the art. Generally, a labelled probe that is complementary to a nucleic acid sequence suspected of being in a sample is prepared. Preferably, the target nucleic acid molecule is immobilized. The presence of probe hybridized to the target nucleic acid molecule indicates the presence of the nucleic acid molecule in the sample. Examples of suitable methods are described by Dallas et al. in “The Characterization of an Escherichia Coli Plasmid Determinant that Encodes for the Production of a Heat-labile Enterotoxin.” in K. N. Timmis and A. Puehler, eds, Plasmids of Medical, Environmental, and Commercial Importance, Elsevier/North-Holland Publishing Co., Amsterdam, pages 113-122 (1975); Grunstein and Hogness in Proc. Natl. Acad. Sci USA 72, 3961-3965 (1975); Palva et al. in U.S. Pat. No. 4,731,325, which is assigned to Orion-yhtyma, Espoo, Finland; Mullis et al. in U.S. Pat. No. 4,683,195, which is assigned to Cetus Corporation, Emeryville, Calif.; Schneider et al. in U.S. Pat. No. 4,882,269, which is assigned to Princeton University, and Segev in PCT Application WO 90/01069. The Schneider et al. patent and the Segev application are both licensed to ImClone Systems Inc., New York City.

[0101] The probes described above are labelled in accordance with methods known in the art. Methods for labelling oligonucleotide probes have been described, for example, by Leary et al, Proc. Natl. Acad. Sci. USA (1983) 80:4045; Renz and Kurz, Nucl. Acids Res. (1984) 12:3435; Richardson and Gumport, Nucl. Acids Res. (1983) 11:6167; Smith et al, Nucl. Acids Res. (1985) 13:2399; and Meinkoth and Wahl, Anal. Biochem. (1984) 138:267.

EXAMPLES

[0102] 1. Bacterial Strains and Culture Conditions.

[0103] Gonococcal strain FA19 is passed from frozen stock once on GCB agar and then used to inoculate flasks containing 1 liter of GCB broth to a starting density of 20 KU (Klett units). The culture is grown with 5% CO₂ at 37° C. with vigorous shaking until reaching a density of 40 KU at which time the chelator, desferal, is added to a final concentration of 50 μM. Cells are harvested 4 hours after addition.

[0104] Meningococcal strain FAM20 is prepared is the same manner as gonococcal strain FA19, except for the use of Chelex-treated CDM instead of GCB and desferal.

[0105] 2a. Affinity Purification of Gonococcal Transferrin-binding Protein.

[0106] The methods used for the preparation of membranes and isolation and purification of the gonococcal transferrin-binding protein is similar to that of Schryvers and Morris Infect. and Immun. 56, 1144-1149 (1988) for the preparation of meningococcal lactoferrin-binding protein. This method in the paper of Schryvers and Morris is incorporated herein by reference. The following modifications of the method of Schryvers and Morris are introduced. 625 μg of biotinylated transferrin (prepared by the method of Schryvers using Biotin-S-S-NHS from Pierce as the biotinylation reagent) is mixed with 25 mg total membrane protein from gonococcal strain FA19 in 25 ml of 100 mM NaCl/50 mM Tris, pH 8.0. The mixture is incubated at room temperature for 1 hour with gentle agitation. The membranes are pelletted at 17,000×g for 10 minutes. Pellets are resuspended in 25 ml of 100 mM NaCl/50 mM Tris, pH 8.0, followed by addition of NA₂EDTA to a final concentration of 10 mM and N-lauroyl-sarcosine to a final concentration of 0.75%. Membranes are solubilized for 10 minutes at room temperature with agitation. 2.5 ml of streptavidin-agarose (Sigma) is added and is allowed to bind for 1 hour at room temperature. The resin is spun out at 3000×g for 5 minutes, the supernatant is removed and the resin is washed twice in 1M NaCl/50 mM Tris, pH 8.0 with 5 mM EDTA and 0.5% N-lauroyl-sarcosine and then twice in 1M NaCl/50 mM Tris, pH 8.0 with no additions. Protein is eluted from the matrix with 0.45% N-lauroyl-sarcosine and 125 mM beta-mercaptoethanol in IM NaCl/50 mM Tris, pH 8.0.

[0107] 2b. Affinity Purification of Meningococcal Transferrin-binding Protein.

[0108] The procedure of example 2a is repeated, except meningococcal strain FAM20 is substituted for gonococcal strain FA19.

[0109] 3a. Isolation of Gonococcal Transferrin-binding Protein.

[0110] The eluate from the affinity preparation (Example 2a) is concentrated using Amicon concentrators (30,000 MW cutoff). The resulting concentrated protein preparation is solubilized in 20% glycerol, 4% SDS, 130 mM Tris, pH 8.0, 10 μg/ml bromophenol blue and separated on a 7.5% SDS polyacrylamide gel according to the method of Laemmli, Nature, 227, 680-685 (1970). The gel is stained with Coomassie Brilliant Blue to visualize the proteins. Two protein species are resolved as single bands by this method. Transferrin has a molecular weight of approximately 80kD. The transferrin-binding protein has a molecular weight of 100 kD. The 100 kD protein band is excised, lyophilized and macerated.

[0111] 3b. Isolation of Meningococcal Transferrin-binding Protein.

[0112] The procedure of example 3A is repeated, except the eluate from example 2b is substituted for the eluate of example 2a.

[0113] 4. Antisera Against the Transferrin-binding Protein.

[0114] The fine powders resulting from examples 3a and 3b are separately resuspended in saline, mixed with an equal volume of Freund's adjuvant (complete for the first injection; incomplete for subsequent injections) and injected into New England White, female rabbits. Injections are spaced two weeks apart. Anti-100 kD protein antibody can be detected two weeks after the third injection by western blotting against purified transferrin-binding protein.

[0115] 5a. Gonococcal DNA Lambda-gt11 Expression Library.

[0116] Chromosomal DNA from gonococcal strain FA19 is isolated according to Seifert et al, J. Bacteriol. 172, 40-46 (1990) and sonicated by standard procedures (Maniatis et al, 1982) to yield an average fragment size of 500 bp. EcoRI linkers are added and the resulting fragments are ligated into EcoRI digested lambda-gt11 DNA (Maniatis et al, 1982). Ligated DNA is packaged using a kit manufactured by Promega.

[0117] 5b. Meningococcal DNA Lambda-gt11 Expression Library.

[0118] Chromosomal DNA from meningococcal strain FAM20 is isolated in accordance with Seifert et al, J. Bacteriol. 172, 40-46 (1990) and digested with the restriction endonuclease HincII. EcoRI linkers are added, and the resultant DNA molecule is digested with EcoRI and ligated into EcoRI digested lambda-Zap (Stratagene). Ligated DNA is packaged using a kit manufactured by Promega.

[0119] 6a. Immunological Screening of the Expression Library.

[0120] Approximately 500,000 plaques obtained from the library of examples 5a and 5b are screened by the immunological screening method described in Stratagene's protocol accompanying the Picoblue Immunological Screening Kit. Briefly, the primary antisera is absorbed with an E. coli/phage lysate available from Stratagene (LaJolla, Calif.) according to their protocol. Approximately 5×10⁴ pfu (plaque forming units) are plated on the E. coli host strain, Y1090. Nitrocellulose filters, soaked in 10 mM isopropylthiogalactoside (IPTG) are laid upon plates following 3-4 hours incubation at 42° C. Plates are then incubated overnight after which filters are removed, washed in tris-buffered saline and 0.05% Tween-20 (TBST) and blocked for one hour in tris-buffered saline and 5% bovine serum albumen. The filters are then incubated with a 1:200 dilution of the absorbed primary antibody for one hour. After incubation with primary antibody, filters are washed extensively with TBST and then incubated with the secondary antibody (1:3000 dilution of goat anti-rabbit antibody conjugated to alkaline phosphatase, purchased from Bio-Rad) for one hour. Filters are then washed extensively with TBST and finally incubated in 0.3 mg/ml nitroblue tetrazolium (NBT), 0.15 mg/ml 5-bromo-4-chloro-3-indoyl phosphate (BCIP), 100 mM Tris pH 9.8, 100 mM NaCl, 5 mM MgCl₂ until sufficient color develops.

[0121] Plaques which bind the transferrin-binding protein specific antisera are picked and purified away from other non-reacting plaques. The DNA from purified phage is isolated and purified using anion-exchange chromatography (column purchased from Qiagen, Studio City, Calif.).

[0122] 6b. Screening the Expression Library with DNA Probes

[0123] Plagues obtained from the library of examples 5a and 5b are also screened using labeled DNA probes. Oligomer TfBP1, 2, 3, or 5 is labeled nonradioactively using digoxigenin-11-dUTP and a DNA tailing kit, both manufactured by Boehringer Mannheim Biochemicals (BMB). The sequences of the oligomers are: TFBP1: GAG CCC GCC AAT GCG CCG CT TFBP2: AGC GGC GCA TTG GCG GGC TC TFBP3: GGG GCG CAT CGG CGG TGC GG TFBP5: AAA ACA GTT GGA TAC CAT AC

[0124] The protocol for DNA labeling and detection are available from BMB with the Genius nonradioactive dna labeling and detecting kit. Alternatively, the same oligomers are labelled radioactively with alpha-32p-dCTP and BMB's DNA tailing kit using standard techniques (Maniatis et al, 1982).

[0125] 7. Amplification and Sequencing of DNA.

[0126] The DNA obtained in example 5 or 6 is amplified by the PCR technique (Sambrook et al, (eds), Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Press (1989)) using lambda-gt11-specific oligomers as amplimers. Inserts thus amplified are cloned into Bluescript vectors (Stratagene) using standard techniques (Maniatis et al, 1982) and sequenced by the dideoxy chain terminating method of Sanger et al, Proc. Natl. Acad. Sci USA 74, 5463-5467 (1977) using Sequenase (United States Biochemical Corp., Cleveland, Ohio). See SEQ ID NO:1.

[0127] 8. Additional Sequence of the 100 kD Transferrin Binding Protein Gene from Gonococcal Strain FA19.

[0128] Using the general methods of examples 6 and 7, a chromosomal Sau3AI fragment of approximately 1.0 kbp is identified. This fragment is cloned into the BamHI site of the vector pHSS6-GCU (Elkins et al. J. Bacteriol, 173, 3911-3913 (1991)). (The GCU designation indicates that a 10 bp sequence, known as the gonococcal uptake sequence, is included in the vector.) This sequence is known to mediate species-specific uptake of DNA into the gonococcus (Elkins et al., Id.). The host strain for this cloning is HB101. The resulting clone is known as pUNCH 403.

[0129] The insert in pUNCH 403 is sequenced in its entirety using double stranded templates prepared according to the method described by Kraft et al. in Biotechniques 6, 554-556 (1988). The sequence is determined by means of Sanger's dideoxy method using Sequenase (United States Biochemicals).

[0130] 9. Evidence of Structure and Function of the 100 kD Transferrin Binding Protein.

[0131] To determine the effect of inactivation of the 100 kD transferrin binding protein gene, transposon insertions are isolated along the length of the insert in pUNCH403 according to the protocol described by Seifert et al. in Genetic Engineering, Principals and Methods, Setlow, J. K. and Holleander, A., eds., Plenum Press, N.Y., Vol. 8, pages 123-134. mTn3CAT transposons are inserted by shuttle mutagenesis in E. coli, and chloramphenicol resistant transformants are then selected in FA19 to create mutants. mTn3CAT transposons are referred to by Seifert et al. as m-Tn3(Cm). Mutants are then scored for their ability to grow on transferrin as their sole iron source and their ability to express the 100 kD protein as assayed by Western blot. The results of that experiment are shown in FIG. 3. Transposons at positions designated “I”, 44, 37, and 24 ablate both expression of the 100 kD protein and its ability to grow on transferrin. The transposon at position “A”, however, allowed some growth on transferrin and the expression of some detectable native length transferrin binding protein. These results confirm the hypothesis that the structural gene encoding the 100 kD protein begins at position 406, since an insertion upstream of this point allows expression of the wild-type length protein. The fact that expression is not detected at wild-type levels in mutant “A” indicates that the region upstream of the putative start codon is important for regulation of the gene encoding the 100 kD protein.

[0132] 10. The construction and Screening of Meningococcal Genomic Library.

[0133] The 95 kD meningococcal transferrin binding protein gene is cloned in three steps. In the first step, using gonococcal anti-100 kD protein antibody, a 1.3 kb HincII/EcoRI fragment from a lambda Zap II (Stratagene) library is identified (see FIG. 4). The antigen used to generate the antibody is described in Example 4. The method for screening the library is described in Example 6a. The 1.3 kb fragment contains about 500 bp of the 95 kd protein structural gene. This clone hybridizes to a single 5 kb ClaI fragment in the meningococcal strain FAM20 chromosome. A partial 5 kb ClaI library in the vector pHSS6-GCU is constructed, and a 5 kb ClaI/ClaI fragment is cloned using the 1.3 kb fragment as a probe. In step 3, a 1.7 kb EcoRI/ClaI fragment (generated from the 5 kb ClaI fragment obtained in step 2) is used as a probe, resulting in the cloning of the adjacent HincII fragment from a lambda Zap II library. This EcoRI/HincII fragment is about 2.0 kb in size. Fragments generated from the 2.0 kb EcoRI/HincII fragment are used as probes to screen the lambda Zap II library, resulting in the clone shown in step 4, which contains the 3′ end of the gene encoding the 95 kd transferrin-binding protein. The fragments shown in steps 1, 3 and 4 are sequenced by generating unidirectional deletions using Exonuclease III and VII as described by E. Ozkaynak and S. D. Putney in Biotechniques 5, 770 (1987). The complete DNA sequence of the structural gene encoding meningococcal TBP1 as determined from these fragments is shown in FIG. 5 and SEQ ID NO. 3.

[0134] 11. Evidence of Structure and Function of the 95 kD Transferrin Binding Protein.

[0135] The 1.3 kb HincII/EcoRI fragment is used to mutagenize the meningococcal 95 kD protein gene. The same shuttle mutagenesis procedure described in example 9 is employed, except that, instead of mTn3CAT transposons, mTn3erm transposons are introduced into the 1.3 kb clone. mTn3erm transposons are made by modifying the mTn3CAT transposons described in example 9 so as to confer erythromycin resistance. This modification permits erythromycin resistant meningococcal transformants to be selected. These transformants are screened for their ability to grow on transferrin plates as described in example 9. Results of this mutagenesis experiments are detailed in FIG. 6. While mTn3erm insertions 1 and 2 completely abolished the expression of the 95 kD protein and the ability of the clones to grown on transferrin plates, mTn3erm insertions 3 and 4 exhibited some growth on transferrin and showed some amount of 95 kD protein on Western blots. Based on the sequencing and mutagenesis data it appears that the mTn3erm insertions 1 and 2 are in the structural gene and promoter region, respectively, while insertions 3 and 4 seem to be in an upstream region that might be involved in the positive regulation of expression.

SUPPLEMENTAL REFERENCES

[0136] The invention as claimed is enabled in accordance with the specification and readily available references and starting materials. Nevertheless, the following cell lines have been deposited in the American Type Culture Collection, Bethesda, Md. on Jul. 16, 1990 in order to facilitate the making and using of the invention:

[0137] Meningococcal cell line FAM18 (Accession Number ATCC₅₅₀₇₁)

[0138] Meningococcal cell line FAM20 (Accession Number ATCC₅₅₀₇₂)

[0139] Gonococcal cell line FA19 (Accession Number ATCC₅₅₀₇₃)

[0140] In addition, the following brochures containing useful protocols and information are available in the file history of this specification.

[0141] “Predigested Lambda Zap/Eco RI Cloning Kit Instruction Manual,” Stratagene, La Jolla, Calif. (Nov. 20, 1987);

[0142] “Gigapack Plus” (for packaging recombinant lambda phage), Stratagene, La Jolla, Calif. (Apr. 25, 1988);

[0143] “picoBlue Immunoscreening Kit” Instruction Manual,” Stratagene, La Jolla, Calif. (May 19, 1989); and

[0144] “Genius Nonradioactive DNA Labeling and Detection Kit,” Boehringer Mannheim Biochemicals, Indianapolis, Ind. (January, 1989).

1 8 3286 base pairs nucleic acid double linear cDNA NO NO N-terminal Neisseria gonorrheae FA19 CDS 406..3150 1 AACCGCTGAA AACAGGTCGG AGGCAACCTT TACCATTGAC GCCATGATTG AGGGCAACGG 60 CTTTAAAGGT ACGGCGAAAA CCGGTAATGA CGGATTTGCG CCGGATCAAA ACAATAGCAC 120 CGTTACACAT AAAGTGCACA TCGCAAATGC CGAAGTGCAG GGCGGTTTTT ACGGGCCTAA 180 CGCCGAAGAG TTGGGCGGAT GGTTTGCCTA TCCGGGCAAT GAACAAACGA AAAATGCAAC 240 AGTTGAATCC GGCAATGGAA ATTCAGCAAG CAGTGCAACT GTCGTATTCG GTGCGAAACG 300 CCAAAAGCTT GTGAAATAAG CACGGCTGCC GAACAATCGA GAATAAGGCT TCAGACGGCA 360 TCGTTCCTTC CGATTCCGTC TGAAAGCGAA GATTAGGGAA ACACT ATG CAA CAG 414 Met Gln Gln 1 CAA CAT TTG TTC CGA TTA AAT ATT TTA TGC CTG TCT TTA ATG ACT GCG 462 Gln His Leu Phe Arg Leu Asn Ile Leu Cys Leu Ser Leu Met Thr Ala 5 10 15 CTG CCC GCT TAT GCA GAA AAT GTG CAA GCC GGA CAA GCA CAG GAA AAA 510 Leu Pro Ala Tyr Ala Glu Asn Val Gln Ala Gly Gln Ala Gln Glu Lys 20 25 30 35 CAG TTG GAT ACC ATA CAG GTA AAA GCC AAA AAA CAG AAA ACC CGC CGC 558 Gln Leu Asp Thr Ile Gln Val Lys Ala Lys Lys Gln Lys Thr Arg Arg 40 45 50 GAT AAC GAA GTA ACC GGT TTG GGC AAA TTG GTC AAA ACC GCC GAC ACC 606 Asp Asn Glu Val Thr Gly Leu Gly Lys Leu Val Lys Thr Ala Asp Thr 55 60 65 CTC AGC AAG GAA CAG GTA CTC GAC ATC CGC GAC CTG ACG CGT TAC GAC 654 Leu Ser Lys Glu Gln Val Leu Asp Ile Arg Asp Leu Thr Arg Tyr Asp 70 75 80 CCC GGC ATC GCC GTC GTC GAA CAG GGG CGC GGC GCA AGC TCG GGC TAC 702 Pro Gly Ile Ala Val Val Glu Gln Gly Arg Gly Ala Ser Ser Gly Tyr 85 90 95 TCG ATA CGC GGT ATG GAC AAA AAC CGC GTC TCC TTG ACG GTG GAC GGC 750 Ser Ile Arg Gly Met Asp Lys Asn Arg Val Ser Leu Thr Val Asp Gly 100 105 110 115 TTG GCG CAA ATA CAG TCC TAC ACC GCG CAG GCG GCA TTG GGC GGG ACG 798 Leu Ala Gln Ile Gln Ser Tyr Thr Ala Gln Ala Ala Leu Gly Gly Thr 120 125 130 AGG ACG GCG GGC AGC AGC GGC GCA ATC AAT GAA ATC GAG TAT GAG AAC 846 Arg Thr Ala Gly Ser Ser Gly Ala Ile Asn Glu Ile Glu Tyr Glu Asn 135 140 145 GTC AAG GCT GTC GAA ATC AGC AAA GGC TCA AAC TCG GTC GAA CAA GGC 894 Val Lys Ala Val Glu Ile Ser Lys Gly Ser Asn Ser Val Glu Gln Gly 150 155 160 AGC GGC GCA TTG GCG GGC TCG GTC GCA TTT CAA ACC AAA ACC GCC GAC 942 Ser Gly Ala Leu Ala Gly Ser Val Ala Phe Gln Thr Lys Thr Ala Asp 165 170 175 GAT GTT ATC GGG GAA GGC AGG CAG TGG GGC ATT CAG AGT AAA ACC GCC 990 Asp Val Ile Gly Glu Gly Arg Gln Trp Gly Ile Gln Ser Lys Thr Ala 180 185 190 195 TAT TCC GGC AAA AAC CGG GGG CTT ACC CAA TCC ATC GCG CTG GCG GGG 1038 Tyr Ser Gly Lys Asn Arg Gly Leu Thr Gln Ser Ile Ala Leu Ala Gly 200 205 210 CGC ATC GGC GGT GCG GAG GCT TTG CTG ATC CGC ACC GGG CGG CAC GCG 1086 Arg Ile Gly Gly Ala Glu Ala Leu Leu Ile Arg Thr Gly Arg His Ala 215 220 225 GGG GAA ATC CGC GCC CAC GAA GCC GCC GGA CGC GGC GTT CAG AGC TTC 1134 Gly Glu Ile Arg Ala His Glu Ala Ala Gly Arg Gly Val Gln Ser Phe 230 235 240 AAC AGG CTG GCG CCG GTT GAT GAC GGC AGC AAG TAC GCC TAT TTC ATC 1182 Asn Arg Leu Ala Pro Val Asp Asp Gly Ser Lys Tyr Ala Tyr Phe Ile 245 250 255 GTT GAA GAA GAA TGC AAA AAC GGG GGT CAC GAA AAG TGT AAA GCG AAT 1230 Val Glu Glu Glu Cys Lys Asn Gly Gly His Glu Lys Cys Lys Ala Asn 260 265 270 275 CCG AAA AAA GAT GTT GTC GGC GAA GAC AAA CGT CAA ACG GTT TCC ACC 1278 Pro Lys Lys Asp Val Val Gly Glu Asp Lys Arg Gln Thr Val Ser Thr 280 285 290 CGA GAC TAC ACG GGC CCC AAC CGC TTC CTC GCC GAT CCG CTT TCA TAC 1326 Arg Asp Tyr Thr Gly Pro Asn Arg Phe Leu Ala Asp Pro Leu Ser Tyr 295 300 305 GAA AGC CGG TCG TGG CTG TTC CGC CCG GGT TTT CGT TTT GAA AAC AAA 1374 Glu Ser Arg Ser Trp Leu Phe Arg Pro Gly Phe Arg Phe Glu Asn Lys 310 315 320 CGG CAC TAC ATC GGC GGC ATA CTC GAA CGC ACG CAA CAA ACT TTC GAC 1422 Arg His Tyr Ile Gly Gly Ile Leu Glu Arg Thr Gln Gln Thr Phe Asp 325 330 335 ACG CGC GAT ATG ACG GTT CCG GCA TTT CTG ACC AAG GCG GTT TTT GAT 1470 Thr Arg Asp Met Thr Val Pro Ala Phe Leu Thr Lys Ala Val Phe Asp 340 345 350 355 GCA AAT CAA AAA CAG GCG GGT TCT TTG CGC GGC AAC GGC AAA TAC GCG 1518 Ala Asn Gln Lys Gln Ala Gly Ser Leu Arg Gly Asn Gly Lys Tyr Ala 360 365 370 GGC AAC CAC AAA TAC GGC GGA CTG TTT ACC AGC GGC GAA AAC AAT GCG 1566 Gly Asn His Lys Tyr Gly Gly Leu Phe Thr Ser Gly Glu Asn Asn Ala 375 380 385 CCG GTG GGC GCG GAA TAC GGT ACG GGC GTG TTT TAC GAC GAG ACG CAC 1614 Pro Val Gly Ala Glu Tyr Gly Thr Gly Val Phe Tyr Asp Glu Thr His 390 395 400 ACC AAA AGC CGC TAC GGT TTG GAA TAT GTC TAT ACC AAT GCC GAT AAA 1662 Thr Lys Ser Arg Tyr Gly Leu Glu Tyr Val Tyr Thr Asn Ala Asp Lys 405 410 415 GAC ACT TGG GCG GAT TAT GCC CGC CTC TCT TAC GAC CGG CAG GGC ATC 1710 Asp Thr Trp Ala Asp Tyr Ala Arg Leu Ser Tyr Asp Arg Gln Gly Ile 420 425 430 435 GGT TTG GAC AAC CAT TTT CAG CAG ACG CAC TGT TCC GCC GAC GGT TCG 1758 Gly Leu Asp Asn His Phe Gln Gln Thr His Cys Ser Ala Asp Gly Ser 440 445 450 GAC AAA TAT TGC CGT CCG AGT GCC GAC AAG CCG TTT TCC TAT TAC AAA 1806 Asp Lys Tyr Cys Arg Pro Ser Ala Asp Lys Pro Phe Ser Tyr Tyr Lys 455 460 465 TCC GAC CGC GTG ATT TAC GGG GAA AGC CAT AAG CTC TTG CAG GCG GCA 1854 Ser Asp Arg Val Ile Tyr Gly Glu Ser His Lys Leu Leu Gln Ala Ala 470 475 480 TTC AAA AAA TCC TTC GAT ACC GCC AAA ATC CGC CAC AAC CTG AGC GTG 1902 Phe Lys Lys Ser Phe Asp Thr Ala Lys Ile Arg His Asn Leu Ser Val 485 490 495 AAT CTC GGT TAC GAC CGC TTC GGC TCT AAT CTC CGC CAT CAG GAT TAT 1950 Asn Leu Gly Tyr Asp Arg Phe Gly Ser Asn Leu Arg His Gln Asp Tyr 500 505 510 515 TAT TAT CAA AGT GCC AAC CGC GCC TAT TCG TTG AAA ACG CCC CCT CAA 1998 Tyr Tyr Gln Ser Ala Asn Arg Ala Tyr Ser Leu Lys Thr Pro Pro Gln 520 525 530 AAC AAC GGC AAA AAA ACC AGC CCC AAC GGC AGA GAA AAG AAT CCC TAT 2046 Asn Asn Gly Lys Lys Thr Ser Pro Asn Gly Arg Glu Lys Asn Pro Tyr 535 540 545 TGG GTC AGC ATA GGC AGG GGA AAT GTC GTT ACG AGG CAA ATC TGC CTC 2094 Trp Val Ser Ile Gly Arg Gly Asn Val Val Thr Arg Gln Ile Cys Leu 550 555 560 TTT GGC AAC AAT ACT TAT ACG GAC TGC ACG CCG CGC AGC ATC AAC GGC 2142 Phe Gly Asn Asn Thr Tyr Thr Asp Cys Thr Pro Arg Ser Ile Asn Gly 565 570 575 AAA AGC TAT TAC GCG GCG GTC CGG GAC AAT GTC CGT TTG GGC AGG TGG 2190 Lys Ser Tyr Tyr Ala Ala Val Arg Asp Asn Val Arg Leu Gly Arg Trp 580 585 590 595 GCG GAT GTC GGC GCG GGC TTG CGC TAC GAC TAC CGC AGC ACG CAT TCG 2238 Ala Asp Val Gly Ala Gly Leu Arg Tyr Asp Tyr Arg Ser Thr His Ser 600 605 610 GAC GAC GGC AGC GTT TCC ACC GGC ACG CAC CGC ACC CTG TCC TGG AAC 2286 Asp Asp Gly Ser Val Ser Thr Gly Thr His Arg Thr Leu Ser Trp Asn 615 620 625 GCC GGC ATC GTC CTC AAA CCT GCC GAC TGG CTG GAT TTG ACT TAC CGC 2334 Ala Gly Ile Val Leu Lys Pro Ala Asp Trp Leu Asp Leu Thr Tyr Arg 630 635 640 ACT TCA ACC GGC TTC CGC CTG CCC TCG TTT GCG GAA ATG TAC GGC TGG 2382 Thr Ser Thr Gly Phe Arg Leu Pro Ser Phe Ala Glu Met Tyr Gly Trp 645 650 655 CGG TCG GGC GAT AAA ATA AAA GCC GTC AAA ATC GAT CCG GAA AAA TCG 2430 Arg Ser Gly Asp Lys Ile Lys Ala Val Lys Ile Asp Pro Glu Lys Ser 660 665 670 675 TTC AAC AAA GAA GCC GGC ATC GTG TTT AAA GGC GAT TTC GGC AAC TTG 2478 Phe Asn Lys Glu Ala Gly Ile Val Phe Lys Gly Asp Phe Gly Asn Leu 680 685 690 GAG GCA AGT TGG TTC AAC AAT GCC TAC CGC GAT TTG ATT GTC CGG GGT 2526 Glu Ala Ser Trp Phe Asn Asn Ala Tyr Arg Asp Leu Ile Val Arg Gly 695 700 705 TAT GAA GCG CAA ATT AAA GAC GGC AAA GAA CAA GTC AAA GGC AAC CCG 2574 Tyr Glu Ala Gln Ile Lys Asp Gly Lys Glu Gln Val Lys Gly Asn Pro 710 715 720 GCT TAC CTC AAT GCC CAA AGC GCG CGG ATT ACC GGC ATC AAT ATT TTG 2622 Ala Tyr Leu Asn Ala Gln Ser Ala Arg Ile Thr Gly Ile Asn Ile Leu 725 730 735 GGC AAA ATC GAT TGG AAC GGC GTA TGG GAT AAA TTG CCC GAA GGT TGG 2670 Gly Lys Ile Asp Trp Asn Gly Val Trp Asp Lys Leu Pro Glu Gly Trp 740 745 750 755 TAT TCC ACA TTT GCC TAT AAT CGT GTC CGT GTC CGC GAC ATC AAA AAA 2718 Tyr Ser Thr Phe Ala Tyr Asn Arg Val Arg Val Arg Asp Ile Lys Lys 760 765 770 CGC GCA GAC CGC ACC GAT ATT CAA TCA CAC CTG TTT GAT GCC ATC CAA 2766 Arg Ala Asp Arg Thr Asp Ile Gln Ser His Leu Phe Asp Ala Ile Gln 775 780 785 CCC TCG CGC TAT GTC GTC GGC TCG GGC TAT GAC CAA CCG GAA GGC AAA 2814 Pro Ser Arg Tyr Val Val Gly Ser Gly Tyr Asp Gln Pro Glu Gly Lys 790 795 800 TGG GGC GTG AAC GGT ATG CTG ACT TAT TCC AAA GCC AAG GAA ATC ACA 2862 Trp Gly Val Asn Gly Met Leu Thr Tyr Ser Lys Ala Lys Glu Ile Thr 805 810 815 GAG TTG TTG GGC AGC CGG GCT TTG CTC AAC GGC AAC AGC CGC AAT ACA 2910 Glu Leu Leu Gly Ser Arg Ala Leu Leu Asn Gly Asn Ser Arg Asn Thr 820 825 830 835 AAA GCC ACC GCG CGC CGT ACC CGC CCT TGG TAT ATT GTG GAC GTG TCC 2958 Lys Ala Thr Ala Arg Arg Thr Arg Pro Trp Tyr Ile Val Asp Val Ser 840 845 850 GGT TAT TAC ACG GTT AAA AAA CAC TTC ACC CTC CGT GCG GGC GTG TAC 3006 Gly Tyr Tyr Thr Val Lys Lys His Phe Thr Leu Arg Ala Gly Val Tyr 855 860 865 AAC CTC CTC AAC CAC CGC TAT GTT ACT TGG GAA AAT GTG CGG CAA ACT 3054 Asn Leu Leu Asn His Arg Tyr Val Thr Trp Glu Asn Val Arg Gln Thr 870 875 880 GCC GCC GGC GCA GTC AAC CAA CAC AAA AAT GTC GGC GTT TAC AAC CGA 3102 Ala Ala Gly Ala Val Asn Gln His Lys Asn Val Gly Val Tyr Asn Arg 885 890 895 TAT GCC GCC CCC GGC CGC AAC TAC ACA TTT AGC TTG GAA ATG AAG TTC 3150 Tyr Ala Ala Pro Gly Arg Asn Tyr Thr Phe Ser Leu Glu Met Lys Phe 900 905 910 915 TAAACGTCCG AACGCCGCAA ATGCCGTCTG AAAGGCTTCA GACGGCGTTT TTTTTACACA 3210 ATCCCCACCG TTTCCCATCC TTCCCGATAC ACCGTAATCC CGAAACCCGT CATTCCCGCG 3270 CAGGCGTGCA TCCGGG 3286 915 amino acids amino acid linear protein 2 Met Gln Gln Gln His Leu Phe Arg Leu Asn Ile Leu Cys Leu Ser Leu 1 5 10 15 Met Thr Ala Leu Pro Ala Tyr Ala Glu Asn Val Gln Ala Gly Gln Ala 20 25 30 Gln Glu Lys Gln Leu Asp Thr Ile Gln Val Lys Ala Lys Lys Gln Lys 35 40 45 Thr Arg Arg Asp Asn Glu Val Thr Gly Leu Gly Lys Leu Val Lys Thr 50 55 60 Ala Asp Thr Leu Ser Lys Glu Gln Val Leu Asp Ile Arg Asp Leu Thr 65 70 75 80 Arg Tyr Asp Pro Gly Ile Ala Val Val Glu Gln Gly Arg Gly Ala Ser 85 90 95 Ser Gly Tyr Ser Ile Arg Gly Met Asp Lys Asn Arg Val Ser Leu Thr 100 105 110 Val Asp Gly Leu Ala Gln Ile Gln Ser Tyr Thr Ala Gln Ala Ala Leu 115 120 125 Gly Gly Thr Arg Thr Ala Gly Ser Ser Gly Ala Ile Asn Glu Ile Glu 130 135 140 Tyr Glu Asn Val Lys Ala Val Glu Ile Ser Lys Gly Ser Asn Ser Val 145 150 155 160 Glu Gln Gly Ser Gly Ala Leu Ala Gly Ser Val Ala Phe Gln Thr Lys 165 170 175 Thr Ala Asp Asp Val Ile Gly Glu Gly Arg Gln Trp Gly Ile Gln Ser 180 185 190 Lys Thr Ala Tyr Ser Gly Lys Asn Arg Gly Leu Thr Gln Ser Ile Ala 195 200 205 Leu Ala Gly Arg Ile Gly Gly Ala Glu Ala Leu Leu Ile Arg Thr Gly 210 215 220 Arg His Ala Gly Glu Ile Arg Ala His Glu Ala Ala Gly Arg Gly Val 225 230 235 240 Gln Ser Phe Asn Arg Leu Ala Pro Val Asp Asp Gly Ser Lys Tyr Ala 245 250 255 Tyr Phe Ile Val Glu Glu Glu Cys Lys Asn Gly Gly His Glu Lys Cys 260 265 270 Lys Ala Asn Pro Lys Lys Asp Val Val Gly Glu Asp Lys Arg Gln Thr 275 280 285 Val Ser Thr Arg Asp Tyr Thr Gly Pro Asn Arg Phe Leu Ala Asp Pro 290 295 300 Leu Ser Tyr Glu Ser Arg Ser Trp Leu Phe Arg Pro Gly Phe Arg Phe 305 310 315 320 Glu Asn Lys Arg His Tyr Ile Gly Gly Ile Leu Glu Arg Thr Gln Gln 325 330 335 Thr Phe Asp Thr Arg Asp Met Thr Val Pro Ala Phe Leu Thr Lys Ala 340 345 350 Val Phe Asp Ala Asn Gln Lys Gln Ala Gly Ser Leu Arg Gly Asn Gly 355 360 365 Lys Tyr Ala Gly Asn His Lys Tyr Gly Gly Leu Phe Thr Ser Gly Glu 370 375 380 Asn Asn Ala Pro Val Gly Ala Glu Tyr Gly Thr Gly Val Phe Tyr Asp 385 390 395 400 Glu Thr His Thr Lys Ser Arg Tyr Gly Leu Glu Tyr Val Tyr Thr Asn 405 410 415 Ala Asp Lys Asp Thr Trp Ala Asp Tyr Ala Arg Leu Ser Tyr Asp Arg 420 425 430 Gln Gly Ile Gly Leu Asp Asn His Phe Gln Gln Thr His Cys Ser Ala 435 440 445 Asp Gly Ser Asp Lys Tyr Cys Arg Pro Ser Ala Asp Lys Pro Phe Ser 450 455 460 Tyr Tyr Lys Ser Asp Arg Val Ile Tyr Gly Glu Ser His Lys Leu Leu 465 470 475 480 Gln Ala Ala Phe Lys Lys Ser Phe Asp Thr Ala Lys Ile Arg His Asn 485 490 495 Leu Ser Val Asn Leu Gly Tyr Asp Arg Phe Gly Ser Asn Leu Arg His 500 505 510 Gln Asp Tyr Tyr Tyr Gln Ser Ala Asn Arg Ala Tyr Ser Leu Lys Thr 515 520 525 Pro Pro Gln Asn Asn Gly Lys Lys Thr Ser Pro Asn Gly Arg Glu Lys 530 535 540 Asn Pro Tyr Trp Val Ser Ile Gly Arg Gly Asn Val Val Thr Arg Gln 545 550 555 560 Ile Cys Leu Phe Gly Asn Asn Thr Tyr Thr Asp Cys Thr Pro Arg Ser 565 570 575 Ile Asn Gly Lys Ser Tyr Tyr Ala Ala Val Arg Asp Asn Val Arg Leu 580 585 590 Gly Arg Trp Ala Asp Val Gly Ala Gly Leu Arg Tyr Asp Tyr Arg Ser 595 600 605 Thr His Ser Asp Asp Gly Ser Val Ser Thr Gly Thr His Arg Thr Leu 610 615 620 Ser Trp Asn Ala Gly Ile Val Leu Lys Pro Ala Asp Trp Leu Asp Leu 625 630 635 640 Thr Tyr Arg Thr Ser Thr Gly Phe Arg Leu Pro Ser Phe Ala Glu Met 645 650 655 Tyr Gly Trp Arg Ser Gly Asp Lys Ile Lys Ala Val Lys Ile Asp Pro 660 665 670 Glu Lys Ser Phe Asn Lys Glu Ala Gly Ile Val Phe Lys Gly Asp Phe 675 680 685 Gly Asn Leu Glu Ala Ser Trp Phe Asn Asn Ala Tyr Arg Asp Leu Ile 690 695 700 Val Arg Gly Tyr Glu Ala Gln Ile Lys Asp Gly Lys Glu Gln Val Lys 705 710 715 720 Gly Asn Pro Ala Tyr Leu Asn Ala Gln Ser Ala Arg Ile Thr Gly Ile 725 730 735 Asn Ile Leu Gly Lys Ile Asp Trp Asn Gly Val Trp Asp Lys Leu Pro 740 745 750 Glu Gly Trp Tyr Ser Thr Phe Ala Tyr Asn Arg Val Arg Val Arg Asp 755 760 765 Ile Lys Lys Arg Ala Asp Arg Thr Asp Ile Gln Ser His Leu Phe Asp 770 775 780 Ala Ile Gln Pro Ser Arg Tyr Val Val Gly Ser Gly Tyr Asp Gln Pro 785 790 795 800 Glu Gly Lys Trp Gly Val Asn Gly Met Leu Thr Tyr Ser Lys Ala Lys 805 810 815 Glu Ile Thr Glu Leu Leu Gly Ser Arg Ala Leu Leu Asn Gly Asn Ser 820 825 830 Arg Asn Thr Lys Ala Thr Ala Arg Arg Thr Arg Pro Trp Tyr Ile Val 835 840 845 Asp Val Ser Gly Tyr Tyr Thr Val Lys Lys His Phe Thr Leu Arg Ala 850 855 860 Gly Val Tyr Asn Leu Leu Asn His Arg Tyr Val Thr Trp Glu Asn Val 865 870 875 880 Arg Gln Thr Ala Ala Gly Ala Val Asn Gln His Lys Asn Val Gly Val 885 890 895 Tyr Asn Arg Tyr Ala Ala Pro Gly Arg Asn Tyr Thr Phe Ser Leu Glu 900 905 910 Met Lys Phe 915 3537 base pairs nucleic acid double linear cDNA NO NO N-terminal Neisseria meningitidis FAM18, FAM20, B16B6, group X and group W135 CDS 721..3450 mat-peptide 793..3447 3 GAATTCCGAC GGAGTGGAGC TTTCACTGCT GCCGTCTGAG GGCAATAAGG CGGCATTTCA 60 GCACGAGATT GAGCAAAACG GCGTGAAGGC AACGGTGTGT TGTTCCAACT TGGATTACAT 120 GAGTTTTGGG AAGCTGTCAA AAGAAAATAA AGACGATATG TTCCTGCAAG GTGTCCGCAC 180 TCCAGTATCC GATGTGGCGG CAAGGACGGA GCAAACGCCA AATATCGCGG TACTTGGTAC 240 GGATATATTG CCAACGGCAC AAGCTGGAGC GCGAAGCCTC CAATCAGGAA GGTGGTAATA 300 GGGCAGAGTT TGACGTGGAT TTTTCCACTA AAAAAATCAG TGGCACACTG ACGGCAAAAG 360 ACCGTACGTC TCCTGCGTTT ACTATTACTG CCATGATTAA GGACAACGGT TTTTCAGGTG 420 TGGCGAAAAC CGGTGAAAAC GGCTTTGCGC TGGATCCGCA AAATACCGGA AATTCCCACT 480 ATACGCATAT TGAAGCCACT GTATCCGGCG GTTTCTACGG CAAAAACGCC ATCGAGATGG 540 CGGATCGTTC TCATTTCCGG GAAATGCACC AGAGGGAAAA CAAGAAAAAG CATCGGTGGT 600 ATTCGGTCGG AAACGCCAAC AGCTTGTGCA ATAAGCACGG CTGCCGAACA ATCGAGAATA 660 AGGCTTCAGA CGGCACCGTT CCTTCCGATG CCGTCTGAAA GCGAAGATTA GGGAAACACT 720 ATG CAA CAG CAA CAT TTG TTC CGA TTA AAT ATT TTA TGC CTG TCT TTA 768 Met Gln Gln Gln His Leu Phe Arg Leu Asn Ile Leu Cys Leu Ser Leu -24 -20 -15 -10 ATG ACC GCG CTG CCC GTT TAT GCA GAA AAT GTG CAA GCC GAA CAA GCA 816 Met Thr Ala Leu Pro Val Tyr Ala Glu Asn Val Gln Ala Glu Gln Ala -5 1 5 CAG GAA AAA CAG TTG GAT ACC ATA CAG GTA AAA GCC AAA AAA CAG AAA 864 Gln Glu Lys Gln Leu Asp Thr Ile Gln Val Lys Ala Lys Lys Gln Lys 10 15 20 ACC CGC CGC GAT AAC GAA GTA ACC GGG CTG GGC AAG TTG GTC AAG TCT 912 Thr Arg Arg Asp Asn Glu Val Thr Gly Leu Gly Lys Leu Val Lys Ser 25 30 35 40 TCC GAT ACG CTA AGT AAA GAA CAG GTT TTG AAT ATC CGA GAC CTG ACC 960 Ser Asp Thr Leu Ser Lys Glu Gln Val Leu Asn Ile Arg Asp Leu Thr 45 50 55 CGT TAT GAT CCG GGT ATT GCC GTG GTC GAA CAG GGT CGG GGC GCA AGT 1008 Arg Tyr Asp Pro Gly Ile Ala Val Val Glu Gln Gly Arg Gly Ala Ser 60 65 70 TCC GGC TAT TCA ATA CGC GGC ATG GAT AAA AAC CGC GTT TCC TTA ACG 1056 Ser Gly Tyr Ser Ile Arg Gly Met Asp Lys Asn Arg Val Ser Leu Thr 75 80 85 GTA GAC GGC GTT TCG CAA ATA CAG TCC TAC ACC GCG CAG GCG GCA TTG 1104 Val Asp Gly Val Ser Gln Ile Gln Ser Tyr Thr Ala Gln Ala Ala Leu 90 95 100 GGT GGG ACG AGG ACG GCG GGT AGC AGC GGC GCA ATC AAT GAA ATC GAG 1152 Gly Gly Thr Arg Thr Ala Gly Ser Ser Gly Ala Ile Asn Glu Ile Glu 105 110 115 120 TAT GAA AAC GTC AAG GCC GTT GAA ATC AGC AAG GGT TCG AAT TCA TCA 1200 Tyr Glu Asn Val Lys Ala Val Glu Ile Ser Lys Gly Ser Asn Ser Ser 125 130 135 GAA TAC GGA AAC GGC GCA TTG GCA GGT TCG GTC GCA TTT CAA ACC AAA 1248 Glu Tyr Gly Asn Gly Ala Leu Ala Gly Ser Val Ala Phe Gln Thr Lys 140 145 150 ACC GCA GCC GAC ATT ATC GGA GAG GGA AAA CAG TGG GGC ATT CAG AGT 1296 Thr Ala Ala Asp Ile Ile Gly Glu Gly Lys Gln Trp Gly Ile Gln Ser 155 160 165 AAA ACT GCC TAT TCG GGA AAA GAC CAT GCC CTG ACG CAA TCC CTT GCG 1344 Lys Thr Ala Tyr Ser Gly Lys Asp His Ala Leu Thr Gln Ser Leu Ala 170 175 180 CTT GCC GGA CGC AGC GGC GGC GCG GAA GCC CTC CTT ATT TAT ACT AAA 1392 Leu Ala Gly Arg Ser Gly Gly Ala Glu Ala Leu Leu Ile Tyr Thr Lys 185 190 195 200 CGG CGG GGT CGG GAA ATC CAT GCG CAT AAA GAT GCC GGC AAG GGT GTG 1440 Arg Arg Gly Arg Glu Ile His Ala His Lys Asp Ala Gly Lys Gly Val 205 210 215 CAG AGC TTC AAC CGG CTG GTG TTG GAC GAG GAC AAG AAG GAG GGT GGC 1488 Gln Ser Phe Asn Arg Leu Val Leu Asp Glu Asp Lys Lys Glu Gly Gly 220 225 230 AGT CAG TCA GAT ATT TCA TTG TGC GAA GAA GAA TGC CAC AAT GGA TAT 1536 Ser Gln Ser Asp Ile Ser Leu Cys Glu Glu Glu Cys His Asn Gly Tyr 235 240 245 GCG GCC TGT AAA AAC AAG CTG AAA GAA GAT GCC TCG GTC AAA GAT GAG 1584 Ala Ala Cys Lys Asn Lys Leu Lys Glu Asp Ala Ser Val Lys Asp Glu 250 255 260 CGC AAA ACC GTC AGC ACG CAG GAT TAT ACC GGC TCC AAC CGC TTA CTT 1632 Arg Lys Thr Val Ser Thr Gln Asp Tyr Thr Gly Ser Asn Arg Leu Leu 265 270 275 280 GCG AAC CCG CTT GAG TAT GGC AGC CAA TCA TGG CTG TTC CGA CCG GGT 1680 Ala Asn Pro Leu Glu Tyr Gly Ser Gln Ser Trp Leu Phe Arg Pro Gly 285 290 295 TGG CAT TTG GAC AAC CGC CAT TAT GTC GGA GCC GTT CTC GAA CGT ACG 1728 Trp His Leu Asp Asn Arg His Tyr Val Gly Ala Val Leu Glu Arg Thr 300 305 310 CAG CAG ACC TTT GAT ACA CGG GAT ATG ACT GTT CCT GCC TAT TTT ACC 1776 Gln Gln Thr Phe Asp Thr Arg Asp Met Thr Val Pro Ala Tyr Phe Thr 315 320 325 AGT GAA GAT TAT GTA CCC GGT TCG CTG AAA GGT CTT GGC AAA TAT TCG 1824 Ser Glu Asp Tyr Val Pro Gly Ser Leu Lys Gly Leu Gly Lys Tyr Ser 330 335 340 GGC GAT AAT AAG GCA GAA AGG CTG TTT GTT CAG GGA GAG GGC AGT ACA 1872 Gly Asp Asn Lys Ala Glu Arg Leu Phe Val Gln Gly Glu Gly Ser Thr 345 350 355 360 TTG CAG GGT ATC GGT TAC GGT ACC GGC GTG TTT TAT GAT GAA CGC CAT 1920 Leu Gln Gly Ile Gly Tyr Gly Thr Gly Val Phe Tyr Asp Glu Arg His 365 370 375 ACT AAA AAC CGC TAC GGG GTC GAA TAT GTT TAC CAT AAT GCT GAT AAG 1968 Thr Lys Asn Arg Tyr Gly Val Glu Tyr Val Tyr His Asn Ala Asp Lys 380 385 390 GAT ACC TGG GCC GAT TAC GCC CGA CTT TCT TAT GAC CGG CAA GGT ATA 2016 Asp Thr Trp Ala Asp Tyr Ala Arg Leu Ser Tyr Asp Arg Gln Gly Ile 395 400 405 GAT TTG GAC AAC CGT TTG CAG CAG ACG CAT TGC TCT CAC GAC GGT TCG 2064 Asp Leu Asp Asn Arg Leu Gln Gln Thr His Cys Ser His Asp Gly Ser 410 415 420 GAT AAA AAT TGC CGT CCC GAC GGC AAT AAA CCG TAT TCT TTC TAT AAA 2112 Asp Lys Asn Cys Arg Pro Asp Gly Asn Lys Pro Tyr Ser Phe Tyr Lys 425 430 435 440 TCC GAC CGG ATG ATT TAT GAA GAA AGC CGA AAC CTG TTC CAA GCA GTA 2160 Ser Asp Arg Met Ile Tyr Glu Glu Ser Arg Asn Leu Phe Gln Ala Val 445 450 455 TTT AAA AAG GCA TTT GAT ACG GCC AAA ATC CGT CAC AAT TTG AGT ATC 2208 Phe Lys Lys Ala Phe Asp Thr Ala Lys Ile Arg His Asn Leu Ser Ile 460 465 470 AAT CTA GGG TAC GAC CGC TTT AAG TCG CAA TTG TCC CAC AGC GAT TAT 2256 Asn Leu Gly Tyr Asp Arg Phe Lys Ser Gln Leu Ser His Ser Asp Tyr 475 480 485 TAT CTT CAA AAC GCA GTT CAG GCA TAT GAT TTG ATA ACC CCG AAA AAG 2304 Tyr Leu Gln Asn Ala Val Gln Ala Tyr Asp Leu Ile Thr Pro Lys Lys 490 495 500 CCT CCG TTT CCC AAC GGA AGC AAA GAC AAC CCG TAT AGG GTG TCT ATC 2352 Pro Pro Phe Pro Asn Gly Ser Lys Asp Asn Pro Tyr Arg Val Ser Ile 505 510 515 520 GGC AAG ACC ACG GTC AAT ACA TCG CCG ATA CCT GGT TTC GGC AAT AAC 2400 Gly Lys Thr Thr Val Asn Thr Ser Pro Ile Pro Gly Phe Gly Asn Asn 525 530 535 ACC TAT ACA GAC TGC ACA CCG AGG AAT ATC GGC GGC AAC GGT TAT TAT 2448 Thr Tyr Thr Asp Cys Thr Pro Arg Asn Ile Gly Gly Asn Gly Tyr Tyr 540 545 550 GCA GCC GTT CAA GAC AAT GTC CGT TTG GGC AGG TGG GCG GAT GTC GGA 2496 Ala Ala Val Gln Asp Asn Val Arg Leu Gly Arg Trp Ala Asp Val Gly 555 560 565 GCA GGC ATA CGT TAC GAT TAC CGC AGC ACG CAT TCG GAA GAT AAG AGT 2544 Ala Gly Ile Arg Tyr Asp Tyr Arg Ser Thr His Ser Glu Asp Lys Ser 570 575 580 GTC TCT ACC GGC ACT CAC CGC AAC CTT TCT TGG AAC GCG GGC GTA GTC 2592 Val Ser Thr Gly Thr His Arg Asn Leu Ser Trp Asn Ala Gly Val Val 585 590 595 600 CTC AAA CCT TTC ACC TGG ATG GAT TTG ACT TAT CGC GCT TCT ACG GGC 2640 Leu Lys Pro Phe Thr Trp Met Asp Leu Thr Tyr Arg Ala Ser Thr Gly 605 610 615 TTC CGT CTG CCG TCG TTT GCC GAA ATG TAT GGC TGG AGA GCC GGG GAG 2688 Phe Arg Leu Pro Ser Phe Ala Glu Met Tyr Gly Trp Arg Ala Gly Glu 620 625 630 TCT TTG AAA ACG TTG GAT CTG AAA CCG GAA AAA TCC TTT AAT AGA GAG 2736 Ser Leu Lys Thr Leu Asp Leu Lys Pro Glu Lys Ser Phe Asn Arg Glu 635 640 645 GCA GGT ATT GTA TTT AAA GGG GAC TTC GGC AAT TTG GAA GCC AGC TAT 2784 Ala Gly Ile Val Phe Lys Gly Asp Phe Gly Asn Leu Glu Ala Ser Tyr 650 655 660 TTC AAC AAT GCC TAT CGC GAC CTG ATT GCA TTC GGT TAT GAA ACC CGA 2832 Phe Asn Asn Ala Tyr Arg Asp Leu Ile Ala Phe Gly Tyr Glu Thr Arg 665 670 675 680 ACT CAA AAC GGG CAA ACT TCG GCT TCT GGC GAC CCC GGA TAC CGA AAT 2880 Thr Gln Asn Gly Gln Thr Ser Ala Ser Gly Asp Pro Gly Tyr Arg Asn 685 690 695 GGC CCA AAA TGC ACG GTA GTA GCC GGT ATC AAT ATT TTG GGT AAA ATC 2928 Gly Pro Lys Cys Thr Val Val Ala Gly Ile Asn Ile Leu Gly Lys Ile 700 705 710 GAT TGG CAC GGC GTA TGG GGC GGG TTG CCG GAC GGG TTG TAT TCC ACG 2976 Asp Trp His Gly Val Trp Gly Gly Leu Pro Asp Gly Leu Tyr Ser Thr 715 720 725 CTT GCC TAT AAC CGT ATC AAG GTC AAA GAT GCC GAT ATA CGC GCC GAC 3024 Leu Ala Tyr Asn Arg Ile Lys Val Lys Asp Ala Asp Ile Arg Ala Asp 730 735 740 AGG ACG TTT GTA ACT TCA TAT CTC TTT GAT GCC GTC CAA CCT TCA CGA 3072 Arg Thr Phe Val Thr Ser Tyr Leu Phe Asp Ala Val Gln Pro Ser Arg 745 750 755 760 TAT GTA TTG GGT TTG GGT TAC GAC CAT CCT GAC GGA ATA TGG GGC ATC 3120 Tyr Val Leu Gly Leu Gly Tyr Asp His Pro Asp Gly Ile Trp Gly Ile 765 770 775 AAT ACG ATG TTT ACT TAT TCC AAG GCA AAA TCT GTT GAC GAA CTG CTC 3168 Asn Thr Met Phe Thr Tyr Ser Lys Ala Lys Ser Val Asp Glu Leu Leu 780 785 790 GGC AGC CAG GCG CTG TTG AAC GGT AAT GCC AAT GCT AAA AAA GCA GCA 3216 Gly Ser Gln Ala Leu Leu Asn Gly Asn Ala Asn Ala Lys Lys Ala Ala 795 800 805 TCA CGG CGG ACG CGG CCT TGG TAT GTT ACG GAT GTT TCC GGA TAT TAC 3264 Ser Arg Arg Thr Arg Pro Trp Tyr Val Thr Asp Val Ser Gly Tyr Tyr 810 815 820 AAT ATC AAG AAA CAC CTG ACC CTG CGC GCA GGT GTG TAC AAC CTC CTC 3312 Asn Ile Lys Lys His Leu Thr Leu Arg Ala Gly Val Tyr Asn Leu Leu 825 830 835 840 AAC TAC CGC TAT GTT ACT TGG GAA AAT GTG CGG CAA ACT GCC GGC GGC 3360 Asn Tyr Arg Tyr Val Thr Trp Glu Asn Val Arg Gln Thr Ala Gly Gly 845 850 855 GCA GTC AAC CAA CAC AAA AAT GTC GGC GTT TAC AAC CGA TAT GCC GCC 3408 Ala Val Asn Gln His Lys Asn Val Gly Val Tyr Asn Arg Tyr Ala Ala 860 865 870 CCC GGC CGA AAC TAC ACA TTT AGC TTG GAA ATG AAG TTT TAAACGTCCA 3457 Pro Gly Arg Asn Tyr Thr Phe Ser Leu Glu Met Lys Phe 875 880 885 AACGCCGCAA ATGCCGTCTG AAAGGCTTCA GACGGCATTT TTTACACAAT TCCCACCGTT 3517 TCCCATCATC CCCGATACAC 3537 909 amino acids amino acid linear protein 4 Met Gln Gln Gln His Leu Phe Arg Leu Asn Ile Leu Cys Leu Ser Leu -24 -20 -15 -10 Met Thr Ala Leu Pro Val Tyr Ala Glu Asn Val Gln Ala Glu Gln Ala -5 1 5 Gln Glu Lys Gln Leu Asp Thr Ile Gln Val Lys Ala Lys Lys Gln Lys 10 15 20 Thr Arg Arg Asp Asn Glu Val Thr Gly Leu Gly Lys Leu Val Lys Ser 25 30 35 40 Ser Asp Thr Leu Ser Lys Glu Gln Val Leu Asn Ile Arg Asp Leu Thr 45 50 55 Arg Tyr Asp Pro Gly Ile Ala Val Val Glu Gln Gly Arg Gly Ala Ser 60 65 70 Ser Gly Tyr Ser Ile Arg Gly Met Asp Lys Asn Arg Val Ser Leu Thr 75 80 85 Val Asp Gly Val Ser Gln Ile Gln Ser Tyr Thr Ala Gln Ala Ala Leu 90 95 100 Gly Gly Thr Arg Thr Ala Gly Ser Ser Gly Ala Ile Asn Glu Ile Glu 105 110 115 120 Tyr Glu Asn Val Lys Ala Val Glu Ile Ser Lys Gly Ser Asn Ser Ser 125 130 135 Glu Tyr Gly Asn Gly Ala Leu Ala Gly Ser Val Ala Phe Gln Thr Lys 140 145 150 Thr Ala Ala Asp Ile Ile Gly Glu Gly Lys Gln Trp Gly Ile Gln Ser 155 160 165 Lys Thr Ala Tyr Ser Gly Lys Asp His Ala Leu Thr Gln Ser Leu Ala 170 175 180 Leu Ala Gly Arg Ser Gly Gly Ala Glu Ala Leu Leu Ile Tyr Thr Lys 185 190 195 200 Arg Arg Gly Arg Glu Ile His Ala His Lys Asp Ala Gly Lys Gly Val 205 210 215 Gln Ser Phe Asn Arg Leu Val Leu Asp Glu Asp Lys Lys Glu Gly Gly 220 225 230 Ser Gln Ser Asp Ile Ser Leu Cys Glu Glu Glu Cys His Asn Gly Tyr 235 240 245 Ala Ala Cys Lys Asn Lys Leu Lys Glu Asp Ala Ser Val Lys Asp Glu 250 255 260 Arg Lys Thr Val Ser Thr Gln Asp Tyr Thr Gly Ser Asn Arg Leu Leu 265 270 275 280 Ala Asn Pro Leu Glu Tyr Gly Ser Gln Ser Trp Leu Phe Arg Pro Gly 285 290 295 Trp His Leu Asp Asn Arg His Tyr Val Gly Ala Val Leu Glu Arg Thr 300 305 310 Gln Gln Thr Phe Asp Thr Arg Asp Met Thr Val Pro Ala Tyr Phe Thr 315 320 325 Ser Glu Asp Tyr Val Pro Gly Ser Leu Lys Gly Leu Gly Lys Tyr Ser 330 335 340 Gly Asp Asn Lys Ala Glu Arg Leu Phe Val Gln Gly Glu Gly Ser Thr 345 350 355 360 Leu Gln Gly Ile Gly Tyr Gly Thr Gly Val Phe Tyr Asp Glu Arg His 365 370 375 Thr Lys Asn Arg Tyr Gly Val Glu Tyr Val Tyr His Asn Ala Asp Lys 380 385 390 Asp Thr Trp Ala Asp Tyr Ala Arg Leu Ser Tyr Asp Arg Gln Gly Ile 395 400 405 Asp Leu Asp Asn Arg Leu Gln Gln Thr His Cys Ser His Asp Gly Ser 410 415 420 Asp Lys Asn Cys Arg Pro Asp Gly Asn Lys Pro Tyr Ser Phe Tyr Lys 425 430 435 440 Ser Asp Arg Met Ile Tyr Glu Glu Ser Arg Asn Leu Phe Gln Ala Val 445 450 455 Phe Lys Lys Ala Phe Asp Thr Ala Lys Ile Arg His Asn Leu Ser Ile 460 465 470 Asn Leu Gly Tyr Asp Arg Phe Lys Ser Gln Leu Ser His Ser Asp Tyr 475 480 485 Tyr Leu Gln Asn Ala Val Gln Ala Tyr Asp Leu Ile Thr Pro Lys Lys 490 495 500 Pro Pro Phe Pro Asn Gly Ser Lys Asp Asn Pro Tyr Arg Val Ser Ile 505 510 515 520 Gly Lys Thr Thr Val Asn Thr Ser Pro Ile Pro Gly Phe Gly Asn Asn 525 530 535 Thr Tyr Thr Asp Cys Thr Pro Arg Asn Ile Gly Gly Asn Gly Tyr Tyr 540 545 550 Ala Ala Val Gln Asp Asn Val Arg Leu Gly Arg Trp Ala Asp Val Gly 555 560 565 Ala Gly Ile Arg Tyr Asp Tyr Arg Ser Thr His Ser Glu Asp Lys Ser 570 575 580 Val Ser Thr Gly Thr His Arg Asn Leu Ser Trp Asn Ala Gly Val Val 585 590 595 600 Leu Lys Pro Phe Thr Trp Met Asp Leu Thr Tyr Arg Ala Ser Thr Gly 605 610 615 Phe Arg Leu Pro Ser Phe Ala Glu Met Tyr Gly Trp Arg Ala Gly Glu 620 625 630 Ser Leu Lys Thr Leu Asp Leu Lys Pro Glu Lys Ser Phe Asn Arg Glu 635 640 645 Ala Gly Ile Val Phe Lys Gly Asp Phe Gly Asn Leu Glu Ala Ser Tyr 650 655 660 Phe Asn Asn Ala Tyr Arg Asp Leu Ile Ala Phe Gly Tyr Glu Thr Arg 665 670 675 680 Thr Gln Asn Gly Gln Thr Ser Ala Ser Gly Asp Pro Gly Tyr Arg Asn 685 690 695 Gly Pro Lys Cys Thr Val Val Ala Gly Ile Asn Ile Leu Gly Lys Ile 700 705 710 Asp Trp His Gly Val Trp Gly Gly Leu Pro Asp Gly Leu Tyr Ser Thr 715 720 725 Leu Ala Tyr Asn Arg Ile Lys Val Lys Asp Ala Asp Ile Arg Ala Asp 730 735 740 Arg Thr Phe Val Thr Ser Tyr Leu Phe Asp Ala Val Gln Pro Ser Arg 745 750 755 760 Tyr Val Leu Gly Leu Gly Tyr Asp His Pro Asp Gly Ile Trp Gly Ile 765 770 775 Asn Thr Met Phe Thr Tyr Ser Lys Ala Lys Ser Val Asp Glu Leu Leu 780 785 790 Gly Ser Gln Ala Leu Leu Asn Gly Asn Ala Asn Ala Lys Lys Ala Ala 795 800 805 Ser Arg Arg Thr Arg Pro Trp Tyr Val Thr Asp Val Ser Gly Tyr Tyr 810 815 820 Asn Ile Lys Lys His Leu Thr Leu Arg Ala Gly Val Tyr Asn Leu Leu 825 830 835 840 Asn Tyr Arg Tyr Val Thr Trp Glu Asn Val Arg Gln Thr Ala Gly Gly 845 850 855 Ala Val Asn Gln His Lys Asn Val Gly Val Tyr Asn Arg Tyr Ala Ala 860 865 870 Pro Gly Arg Asn Tyr Thr Phe Ser Leu Glu Met Lys Phe 875 880 885 20 base pairs nucleic acid single linear DNA (genomic) NO 5 GAGCCCGCCA ATGCGCCGCT 20 20 base pairs nucleic acid single linear DNA (genomic) NO 6 AGCGGCGCAT TGGCGGGCTC 20 20 base pairs nucleic acid single linear DNA (genomic) NO 7 GGGGCGCATC GGCGGTGCGG 20 20 base pairs nucleic acid single linear DNA (genomic) NO 8 AAAACAGTTG GATACCATAC 20 

1. An iron-regulated protein found in Neisseria gonorrhoeae or Neisseria meningitidis outer membranes, wherein the protein is substantially free of: (a) detergent; (b) nitrocellulose/cellulose acetate paper; and (c) other iron-regulated proteins from Neisseria gonorrhoeae and Neisseria meningitidis; and wherein the protein is isolatable by means of a transferrin affinity column; and wherein the protein binds specifically to antisera raised against an iron-regulated outer membrane protein found in Neisseria gonorrhoeae having a molecular weight of approximately 100 kD; and wherein the protein is important in transferrin receptor function in Neisseria gonorrhoeae or Neisseria meningitidis; and functional analogs of such proteins.
 2. The protein of claim 1 wherein the protein is found in Neisseria gonorrhoeae and has a molecular weight of 100 kD.
 3. The protein of claim 1 wherein the protein is found in Neisseria meningitidis and has a molecular weight of 95 kD.
 4. The protein of claim 1 wherein the protein is a Neisseria gonorrhoeae or Neisseria meningitidis transferrin receptor.
 5. The protein of claim 1 wherein the protein has a detectable label.
 6. Isolated antibodies raised against an iron-regulated protein found in Neisseria gonorrhoeae or Neisseria meningitidis outer membranes, wherein the protein is substantially free of: (a) detergent; (b) nitrocellulose/cellulose acetate paper; and (c) other iron-regulated proteins from Neisseria gonorrhoeae and Neisseria meningitidis; and wherein the protein is isolatable by means of a transferrin affinity column; and wherein the protein binds specifically to antisera raised against an iron-regulated outer membrane protein found in Neisseria gonorrhoeae having a molecular weight of approximately 100 kD; and wherein the protein is important in transferrin receptor function in Neisseria gonorrhoeae or Neisseria meningitidis; and functional analogs of such proteins.
 7. The antibodies of claim 6 wherein the antibodies are suitable for detecting the presence of Neisseria gonorrhoeae or Neisseria meningitidis in a sample.
 8. The antibodies of claim 6 wherein the antibodies are suitable for treating a mammal infected with N. gonorrhoeae or N. meningitidis.
 9. The antibodies of claim 6 wherein binding of the antibodies to the protein inhibits growth of Neisseria gonorrhoeae or Neisseria meningitidis.
 10. The antibodies of claim 6 wherein the binding of the antibodies blocks transferrin receptor function of the protein.
 11. The antibodies of claim 6 wherein the antibodies are monoclonal.
 12. A vaccine composition comprising an effective amount of an iron-regulated protein found in Neisseria gonorrhoeae or Neisseria meningitidis outer membranes, wherein the protein is substantially free of: (a) detergent; (b) nitrocellulose/cellulose acetate paper; and (c) other iron-regulated proteins from Neisseria gonorrhoeae and Neisseria meningitidis; wherein the protein is isolatable by means of a transferrin affinity column; wherein the protein binds specifically to antisera raised against an iron-regulated outer membrane protein found in Neisseria gonorrhoeae having a molecular weight of approximately 100 kD; wherein the protein is important in transferrin receptor function in Neisseria gonorrhoeae or Neisseria meningitidis, and functional analogs of such proteins; and a pharmaceutically acceptable carrier.
 13. The vaccine of claim 12 wherein the protein is found in Neisseria gonorrhoeae and has a molecular weight of 100 kD.
 14. The vaccine of claim 12 wherein the protein is found in Neisseria meningitidis and has a molecular weight of 95 kD.
 15. A method of immunizing a mammal against diseases caused by N. gonorrhoeae or N. meningitidis comprising the step of administering to the host a vaccine composition comprising an effective amount of an iron-regulated protein found in Neisseria gonorrhoeae or Neisseria meningitidis outer membranes, wherein the protein is substantially free of: (a) detergent; (b) nitrocellulose/cellulose acetate paper; and (c) other iron-regulated proteins from Neisseria gonorrhoeae and Neisseria meningitidis; wherein the protein is isolatable by means of a transferrin affinity column; wherein the protein binds specifically to antisera raised against an iron-regulated outer membrane protein found in Neisseria gonorrhoeae having a molecular weight of approximately 100 kD; wherein the protein is important in transferrin receptor function in Neisseria gonorrhoeae or Neisseria meningitidis, and functional analogs of such proteins; and a pharmaceutically acceptable carrier.
 16. A method of treating a mammal suffering from diseases caused by N. gonorrhoeae or N. meningitidis, the method comprising administering to the mammal an effective amount of isolated antibodies raised against an iron-regulated protein found in Neisseria gonorrhoeae or Neisseria meningitidis outer membranes, wherein the protein is substantially free of: (a) detergent; (b) nitrocellulose/cellulose acetate paper; and (c) other iron-regulated proteins from Neisseria gonorrhoeae and Neisseria meningitidis; wherein the protein is isolatable by means of a transferrin affinity column; wherein the protein binds specifically to antisera raised against an iron-regulated outer membrane protein found in Neisseria gonorrhoeae having a molecular weight of approximately 100 kD; and wherein the protein is important in transferrin receptor function in Neisseria gonorrhoeae or Neisseria meningitidis; and functional analogs of such proteins.
 17. A nucleotide sequence encoding an iron-regulated protein found in Neisseria gonorrhoeae or Neisseria meningitidis outer membranes, wherein the protein is substantially free of: (a) detergent; (b) nitrocellulose/cellulose acetate paper; and (c) other iron-regulated proteins from Neisseria gonorrhoeae and Neisseria meningitidis; wherein the protein is isolatable by means of a transferrin affinity column; wherein the protein binds specifically to antisera raised against an iron-regulated outer membrane protein found in Neisseria gonorrhoeae having a molecular weight of approximately 100 kD; and wherein the protein is important in transferrin receptor function in Neisseria gonorrhoeae or Neisseria meningitidis; and functional analogs of such proteins and functional derivatives of such proteins.
 18. A method for detecting the presence of antibodies specific for N. gonorrhoeae and N. meningitidis in a sample, the method comprising the steps of: (a) incubating a protein according to claim 1 with a sample suspected of containing antibodies to an iron-regulated protein found in Neisseria gonorrhoeae or Neisseria meningitidis outer membranes, wherein the protein is substantially free of: (1) detergent; (2) nitrocellulose/cellulose acetate paper; and (3) other iron-regulated proteins from Neisseria gonorrhoeae and Neisseria meningitidis; wherein the protein is isolatable by means of a transferrin affinity column; wherein the protein binds specifically to antisera raised against an iron-regulated outer membrane protein found in Neisseria gonorrhoeae having a molecular weight of approximately 100 kD; and wherein the protein is important in transferrin receptor function in Neisseria gonorrhoeae or Neisseria meningitidis; and functional analogs of such proteins; (b) labeling the protein either before, during, or after the incubation of step (a); and (c) detecting the labeled protein bound to an antibody in the sample.
 19. A method for detecting the presence of N. gonorrhoeae or N. meningitidis in a sample comprising the steps of: (a) incubating a sample suspected of containing N. gonorrhoeae or N. meningitidis with an antibody raised against an iron-regulated protein found in Neisseria gonorrhoeae or Neisseria meningitidis outer membranes, wherein the protein is substantially free of: (1) detergent; (2) nitrocellulose/cellulose acetate paper; and (3) other iron-regulated proteins from Neisseria gonorrhoeae and Neisseria meningitidis; wherein the protein is isolatable by means of a transferrin affinity column; wherein the protein binds specifically to antisera raised against an iron-regulated outer membrane protein found in Neisseria gonorrhoeae having a molecular weight of approximately 100 kD; and wherein the protein is important in transferrin receptor function in Neisseria gonorrhoeae or Neisseria meningitidis; and functional analogs of such proteins; (b) labeling the antibody either before, during, or after the incubation of step (a); and (c) detecting the presence of antibody bound to the protein. 